Cholecystokinin b receptor targeting for imaging and therapy

ABSTRACT

Conjugates are described herein where CCK2R targeting ligands are attached to an active moiety, such as therapeutic agent or an imaging agent, through a linker. The conjugates can be used in the detection, diagnosis, imaging and treatment of cancer.

TECHNICAL FIELD

The invention relates to methods of detecting, diagnosing, imaging andtreating cancer in a subject.

BACKGROUND OF INVENTION

The cholecystokinin B receptor (also known as CCK2R, CCKBR, and thegastrin receptor) is a trans-membrane, G protein-coupled receptor. CCK2Ris primarily expressed in (i) the brain and central nervous system, and(ii) the mucosa of the gastrointestinal tract, including parietal andECL cells. It is reported to be most abundantly expressed in thecerebral cortex of the mammalian brain where it has been implicated inthe regulation of memory/learning and response to stress¹. In thealimentary canal, its presence has been detected in the GI tissues, aswell as exocrine and endocrine pancreas. Conflicting reports suggest thereceptor is also expressed in peripheral tissues such as adipocytes andpancreas²⁻⁹, along with CCK1R, a G protein-coupled receptor with 50%homology to CCK2R. The natural ligands of CCK2R are gastrin andcholecystokinin peptides¹.

A number of different types of human tumors have been found thatoverexpress or ectopically express CCK2R in high densities or at highfrequencies including medullary thyroid cancers, insulinomas, small celllung cancers, non small cell lung cancers, bronchial and ilealcarcinoids, GIST tumors, and colon cancers, hepatocellular carcinomas,and pancreatic cancers¹⁰⁻¹⁸. One manner in which disregulation of CCK2Rsignaling may contribute to tumor formation or growth relates to gastrinsignaling through the receptor. Gastrin is a peptide hormone thatstimulates secretion of gastric acid (HCl) by the parietal cells of thestomach upon binding of CCK2R. Gastrin has been reported to be aninhibitor of cancer cell apoptosis, likely through its ability to induceactivation of the serine/threonine protein kinase PKB/Akt¹⁹⁻²¹. Severalreports have shown that inhibiting the CCK2R receptor and the gastrinautocrine loop induces apoptosis and inhibits the proliferation of thecancer cell lines in vivo.

CCK2 receptors found in tumor tissue have been reported to include bothnormal protein as well as a constitutively active a CCK2R-receptorsplice variant (CCK2i4svR) that has the fourth intron inappropriatelyretained, resulting in the addition of 69 amino acids in the thirdintracellular loop domain of the receptor, the domain known to beimportant for signal transduction²⁴⁻²⁹.

Cytotoxic agents that target CCK2R might serve to block uncontrolledactivation of the receptor in tumors. Helpfully, because CCK2R isexpressed in normal brain tissue, the blood brain barrier will blockpolar compounds that could affect normal activity of the receptor in thebrain. Therefore, where CCK2R and its splice variant are expressedoutside of the brain by a tumor, the tumor will be the only tissuetargeted by CCK2R and CCK2i4svR-specific cytotoxic agents.

CCK2R-specific antagonists have been developed and extensivestructure-activity relationships have reported. Both in vitro and invivo studies have demonstrated that the growth potentiating effects ofgastrin can be abolished in the presence of selective CCK2R antagonists.One such antagonist is Z-360, an orally-active CCK2R antagonist (ZeriaPharmaceuticals Co., Ltd., Tokyo, Japan) that has a K_(i)=0.47 nmol/Lwith a selectivity ratio over CCK1R=672. Preclinical studies have shownthat oral administration of Z-360 along with the chemotherapeuticgemcitabine significantly inhibited subcutaneous PANC-1 tumor growthcompared with either agent alone (27.1% inhibition) and significantlyincreased survival compared with the vehicle. This antagonist iscurrently in Phase II human clinical trials for treatment of pancreaticcancer in combination therapy with gemcitabine^(29-31,34-35). The modestanti-tumor effect of the antagonist may be due to several factors.First, many cancers involve multiple genetic mutations that are noteasily treated by a single agent. There exist multiple redundancies,cross talk and possible compensatory mechanisms between signalingpathways. Consequently blocking one part of a pathway may not alwaysprovide enough improvement/antitumor activity that could be translatedto improved survival outcome. Secondly, ongoing mutations in the primarymolecular target of the drug may also result in drug resistance towardssome of these therapies. Third, the presence of a constitutively activesplice variant CCK2i4svR means that blocking CCK2R activity using anantagonist may be less effective.

The development of additional agents that selectively target CCK2R andthat have the ability to deliver payloads (cytotoxic or diagnostic)would broaden the arsenal of agents that could be used in the treatmentand diagnosis of tumors in which CCK2R is expressed. Such agents mayalso be used in the diagnosis and imaging of cancer in a subject.

BRIEF SUMMARY OF INVENTION

The present invention relates to a small group of highly relatedbenzazepine compounds that selectively target and bind CCK2R and thesplice variant CCK2i4svR. The benzazepine compounds are linked to one ormore therapeutic or imaging agents, and these conjugates are used in thetreatment, diagnosis and imaging of tumors expressing CCK2R and thesplice variant.

The invention is thus directed to conjugates comprising a targetingligand linked to an active moiety. The targeting ligand is a benzazepinecompound that selectively binds to CCK2R and/or CCK2i4svR. The activemoiety is a therapeutic agent or imaging agent. The targeting ligand andthe active moiety are joined by a linker as described herein.

In a first embodiment, the invention is directed to conjugatescomprising

B-[L-D]_(n)

wherein B is a targeting ligand, L is a linker, D is an active moiety,and n is an integer of between 1 and 5. In a particular aspect, n=1.

The targeting ligand Z-360 is an excellent example of a targeting ligandthat may be used in the conjugates of the invention.

Additional, non-limiting examples of targeting ligands of the presentinvention are compounds of Formula I

wherein:the bond between X and Y is a single bond or a double bond;R¹=H, CH₃ or heterocycle with R²;R²=heterocycle with R¹, CH₃, CH₂CON(Et)₂, CH₂COC(CH₃)₃, CH₂CONHC(CH₃)₃,

X=CR³ or NR⁴;

R³=phenyl, cyclohexyl, CH₃, CH₂—CH₃

R⁴=phenyl, cyclohexyl,

Y=CH₂, N (dashed=double bond), C═O;

Z=

R⁵=absent, phenyl, Cl, CO₂H, CH₃, OCH₃, NHCH₃, F, SCH₂CO₂H, SCH₂CO₂Et,

and analogs, derivatives, salts and esters thereof.

The active moiety D is a therapeutic agent or an imaging agent.Therapeutic agents include, but are not limited to, radio-therapeuticagents, immunotherapeutic agents, photodynamic therapy agents andchemotherapeutic agents. Imaging agents include, but are not limited to,radio-imaging agents, optical imaging agents, PET imaging agents, MRIcontrast agents, CT contrast agents, and FRET imaging agents. In someaspects, the therapeutic agent is tubulysin B hydrazide or desacetylvinblastine monohydrazide. In some aspects, the imaging agent isfluorescein (FITC), rhodamine, or a cyanine-based near infrared dye suchas 50456, IR800CW, or LS288.

The linker L is a bivalent or polyvalent hydrophilic spacer comprised ofcharged or polar amino acids, sugars or sugar-containing oligomers, or ahydrophilic polymer such as polyethylene glycol. Specific examples ofthe linker are L1, L2 or L3

L1: HN-Glu-Arg-Asp-CO

L2: HN-Glu-PS-Glu-PS-CO

L3: HN-Octanoyl-Glu-PS-Glu-PS-CO

wherein PS is the following formula

In one aspect, the embodiment includes conjugates of the followingformula

B-[L-D]_(n)

wherein B is a targeting ligand of Formula I

wherein:the bond between X and Y is a single bond or a double bond;R¹=H, CH₃ or heterocycle with R²;R²=heterocycle with R¹, CH₃, CH₂CON(Et)₂, CH₂COC(CH₃)₃, CH₂CONHC(CH₃)₃,

X=CR³ or NR⁴;

R³=phenyl, cyclohexyl, CH₃, CH₂—CH₃,

R⁴=phenyl, cyclohexyl,

Y=CH₂, N (dashed=double bond), C═O;

z=

R⁵=absent, phenyl, Cl, CO₂H, CH₃, OCH₃, NHCH₃, F, SCH₂CO₂H, SCH₂CO₂Et,

and analogs, derivatives, salts and esters thereof,

L is a bivalent or polyvalent linker,D is an active moiety, wherein each active moiety is individuallyselected from therapeutic agents and imaging agents, and n is an integerof between 1 and 5.

In a second embodiment, the invention is directed to methods fordetecting a tumor in a subject, comprising administering a conjugate asdefined herein to a subject suspected of having a tumor and detectingthe conjugate in the subject, wherein the active moiety D of theconjugate is an imaging agent.

In this embodiment, imaging agents include, but are not limited to,radio-imaging agents, optical imaging agents, PET imaging agents, MRIcontrast agents, CT contrast agents, and FRET imaging agents. In someaspects, the imaging agent is fluorescein (FITC), rhodamine, S0456,IR800CW, or LS288. In other aspects, the imaging agent is radio-imagingagent comprising ¹¹¹In, ⁹⁹ mTc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga or ⁶⁸Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry,confocal microscopy, fluorescence activated cell sorters, a fluorescenceimaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a third embodiment, the invention is directed to methods fordiagnosing cancer in a subject, comprising administering a conjugate asdefined herein to a subject suspected of having cancer and detecting theconjugate in the subject, wherein the active moiety D of the conjugateis an imaging agent.

In this embodiment, imaging agents include, but are not limited to,radio-imaging agents, optical imaging agents, PET imaging agents, MRIcontrast agents, CT contrast agents, and FRET imaging agents. In someaspects, the imaging agent is fluorescein (FITC), rhodamine, S0456,IR800CW, or LS288. In other aspects, the imaging agent is radio-imagingagent, comprising ¹¹¹In, ⁹⁹mTc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga or ⁶⁸Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry,confocal microscopy, fluorescence activated cell sorters, a fluorescenceimaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a fourth embodiment, the invention is directed to methods for imagingcancer in a subject, comprising administering a conjugate as definedherein to a subject suspected of or having cancer and detecting theconjugate in the subject, wherein the active moiety D of the conjugateis an imaging agent.

In this embodiment, imaging agents include, but are not limited to,radio-imaging agents, optical imaging agents, PET imaging agents, MRIcontrast agents, CT contrast agents, and FRET imaging agents. In someaspects, the imaging agent is fluorescein (FITC), rhodamine, S0456,IR800CW, or LS288. In other aspects, the imaging agent is radio-imagingagent comprising ¹¹¹In, ⁹⁹mTc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga or ⁶⁸Ga.

In this embodiment, the conjugate is detected utilizing flow cytometry,confocal microscopy, fluorescence activated cell sorters, a fluorescenceimaging system, a radioimaging system, MRI, SPECT-CT, or PET imaging.

In a fifth embodiment, the invention is directed to methods for treatinga subject having cancer, comprising administering atherapeutically-effective amount of a conjugate as defined herein to asubject having cancer, wherein the active moiety D of the conjugate is atherapeutic agent.

In this embodiment, therapeutic agents include, but are not limited to,radio-therapeutic agents, immunotherapeutic agents, photodynamic therapyagents and chemotherapeutic agents. In some aspects, the therapeuticagent is tubulysin B hydrazide or desacetyl vinblastine monohydrazide.

In certain aspects of this embodiment, the conjugate will be in apharmaceutical composition comprising the conjugate and apharmaceutically acceptable diluent or carrier.

In relevant embodiments of the invention, cells of the tumor expressCCK2R or CCK2i4svR, or both. The tumor may be, but is not limited to,medullary thyroid cancers, insulinomas, small cell lung cancers, nonsmall cell lung cancers, astrocytoma, gastric cancer, bronchial andileal carcinoids, GIST tumors, and colon cancers, prostate cancer,hepatocellular carcinomas, and pancreatic cancers.

In relevant embodiments of the invention, the cancers that may bediagnosed, imaged or treated include those that express CCK2R orCCK2i4svR, or both. The cancers include, but are not limited to,medullary thyroid cancers, insulinomas, small cell lung cancers, nonsmall cell lung cancers, astrocytoma, gastric cancer, bronchial andileal carcinoids, GIST tumors, and colon cancers, prostate cancer,hepatocellular carcinomas, and pancreatic cancers.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedherein, which form the subject of the claims of the invention. It shouldbe appreciated by those skilled in the art that any conception andspecific embodiment disclosed herein may be readily utilized as a basisfor modifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thatany description, figure, example, etc. is provided for the purpose ofillustration and description only and is by no means intended to definethe limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 graphically displays the binding of CRL-1, CRL-2, CRL-3 and CRL-4to HEK CCK2R tumor cells.

FIG. 2 graphically displays the binding of CRL-2, CRL-3 and CRL-4 to HEKCCKi4svR tumor cells.

FIGS. 3A-B display the overlay of radio-images over white light imagesof mice, injected with CRL-3 ^(99m)Tc displaying the preferentialbinding of CCK2R conjugates to tumor cells.

FIGS. 4A-B are bar diagrams displaying the bio-distribution of CRL-3^(99m)Tc when injected to mice having HEK CCK2R tumors (FIG. 4A) and HEKCCKRi4sv tumors (FIG. 4B).

FIG. 5 contains SPECT-CT images showing the rate of clearance of theCRL-3 ^(99m)Tc from tumor-bearing mice. Mice implanted with HEK CCK2Rtumor xenograft were injected with 5.55 mBq (150 μCi) the CRL-3 ^(99m)Tcand imaged 0.5, 2, 4, and 8 hours post tail vein injection.

FIGS. 6A-B show the binding of conjugate 18 to HEK CCK2R cells (FIG. 6A)and HEK CCKi4svR cells (FIG. 6B). Plates a and e show the binding of 10nM conjugate 18; plates b and f show zoom in of the binding of 10 nMconjugate 19; plates c and g show the binding of 10 nM conjugate18+excess competitor trans illumination; and plates d and h show thebinding of 10 nM conjugate 18+excess competitor.

FIGS. 7A-B are line graphs displaying binding of conjugate 19(CRL-LS288) to HEK CCKRi4sv tumor cells and HEK CCK2R tumor cells,respectively. The solid line represents the cell-bound fluorescence inthe experimental group while the dashed line represents the binding inpresence of free CRL.

FIGS. 8A-C display the overlay of fluorescent imaging of conjugate 19over white light images of mice and their dissected organs, displayingthe preferential binding of the conjugate to CCK2R tumor cells. Tumorsand kidneys are shown with arrows. Dissected organs are labeled asfollows: a-tumor, b-heart, c-lungs, d-spleen, e-liver, f-pancreasg-kidneys, h-small intestines, i-stomach. Mouse a is injected with 10nMols of CRL-LS288+excess CRL whereas mouse b is injected with only 10nmol CRL-LS288

FIGS. 9A-D display the overlay of fluorescent imaging of conjugate 19over white light images of mice and their dissected organs, displayingthe preferential binding of the conjugate to CCK2i4svR tumor cells.Tumors and kidneys are shown with. Dissected organs are labeled asfollows a-tumor, b-heart, c-lungs, d-spleen, e-liver, f-pancreasg-kidneys, h-small intestines, i-stomach. Mouse a is injected with 10nMols of CRL-LS288, whereas mouse b is injected with 10 nmolCRL-LS288+excess CRL and mouse c is a CCK2R negative tumor injected with10 nMols of CRL-LS288.

FIG. 10 shows images of mice with metastatic HEK CCK2i4svR tumorsfollowing intravenous injection of conjugate 19. Images were taken ofthe mouse before (a) and after (b, c, d) various sequential stages oftumor resection guided by the fluorescence of the tumor-targeted NIRdye.

FIG. 11 shows fluorescent (left panel) and color (right panel) images oflungs and heart of tumor-bearing mice shown in FIG. 10.

FIG. 12 shows hematoxylin & eosin staining of fluorescent nodulesresected from a) the lungs, and b) the peritoneal cavity of the tumorbearing mouse shown in FIG. 10. Arrows point to regions containingmalignant disease.

FIGS. 13A-C are line graphs displaying the dose-dependent in vitrocytotoxicity study of compound 23 in HEK CCK2R cells at 2 hours (FIG.13A), 4 hours (FIG. 13B) and 9 hours (FIG. 13C).

FIG. 14 shows in vitro potency of free desacetyl vinblastine hydrazideand conjugate 23 (CRL-desacetyl vinblastine monohydrazide).

FIG. 15 shows in vitro potency of free tubulysin B and conjugate 22(CRL-tubulysin B hydrazide) in the presence and absence of 100 foldZ-360.

FIG. 16 shows in vitro potency of Z-360, untargeted desacetylvinblastine hydrazide (conjugate 25) and untargeted tubulysin Bhydrazide (conjugate 24).

FIGS. 17A-B show the effect of conjugate 23 (CRL-desacetyl vinblastinemonohydrazide) on the a) growth of subcutaneous HEK 293 tumorstransfected with CCK2R and on the b) weights of the treated mice.

FIG. 18 shows H&E staining of mice treated with desacetyl vinblastinehydrazide conjugates: a) untreated group; b) treated with conjugate 23(CRL-desacetyl vinblastine monohydrazide); c) treated with conjugate 23and excess Z-360.

FIG. 19 shows the effect of conjugate 22 (comprising CRL-tubulysin Bhydrazide) on the growth of subcutaneous HEK 293 tumors transfected withCCK2R and on the weights of the treated mice.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

As used herein, “a” or “an” may mean one or more. As used herein whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more. Furthermore, unless otherwise required bycontext, singular terms include pluralities and plural terms include thesingular.

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

Unless otherwise clear by the context, each reference to CCK2R is areference to both CCK2R and CCK2i4svR.

II. Conjugates

The present invention is based on the discovery that useful therapeuticand imaging agents (active moieties) can be joined via a linker tobenzazepine compounds (targeting ligands) that selectively bind CCK2Rand the splice variant CCK2i4svR. Through diligent efforts the inventorsdeveloped linkers and identified locations on the targeting ligands towhich the active moieties could be linked without interfering with theability of the targeting ligands to selectively bind CCK2R andCCK2i4svR. The inventors have thus developed conjugates that comprise atargeting ligand linked to an active moiety, where the targeting ligandis a benzazepine compound that selectively binds to CCK2R and/orCCK2i4svR, and the active moiety is a therapeutic agent or imagingagent.

In particular, the invention is directed to conjugates comprising

B-[L-D]_(n)

wherein B is a targeting ligand, L is a polyvalent linker, D is anactive moiety, and n is an integer of between 1 and 5. These conjugatestarget and bind CCK2R or CCK2i4svR, or both.

The conjugates are targeted to cells that express or over-express CCK2Ror CCK2i4svR through the targeting ligand. Once delivered, theconjugates bind to the receptors. In certain embodiments, the conjugatesremain on the surface of the cell for a period of time sufficient fordetecting, imaging and/or diagnosis. In other embodiments, theconjugates are internalized into the cell by endogenous cellularmechanisms, such as endocytosis, for subsequent detection, imagingand/or diagnosis, or treatment. While attached to the surface or onceinternalized, the conjugates may remain intact or be decomposed,degraded, or otherwise altered to allow the release of the active moietyforming the conjugate. It is appreciated that in detecting, imagingand/or diagnostic configurations, the active moiety may remain attachedto the conjugate or be released either before or after the conjugate hasbeen internalized into the targeted cell. It is further appreciated thatin therapeutic configurations, the active moiety is advantageouslyreleased from the conjugate once it has been internalized into thetargeted cell, or alternatively may be therapeutically active whilestill bound to the targeting ligand.

In a certain aspect, the invention includes conjugates that have abinding constant K_(d) of about 100 nM or less. In another aspect, theconjugates have a K_(d) of about 75 nM or less. In another aspect, theconjugates have a K_(d) of about 50 nM or less. In another aspect, theconjugates have a Kd of about 25 nM or less. In another embodiment, theconjugates described herein exhibit selectivity for CCK2R expressing orCCK2R over-expressing cells or tissues relative to normal tissues suchas blood, lung, liver, spleen, duodenum, skin, muscle, bladder, andprostate, with at least 3-fold selectivity, or at least 5-foldselectivity. In one variation, the conjugates described herein exhibitselectivity for CCK2R expressing or CCK2R over-expressing cells ortissues relative to normal tissues with at least 10-fold selectivity. Itis appreciated that the selectivity observed for imaging is indicativeof the selectivity that may be observed in treating disease statesresponsive to the selective or specific elimination of cells or cellpopulations that express or over-express CCK2R. Based on this dualcapability of CCK2R-targeted conjugates, it is anticipated that aCCK2R-targeted imaging agent may be employed to identify patients thatwill likely respond to a CCK2R-targeted therapeutic agent.

Examples of conjugates of the present invention include CRL-1, CRL-2,CRL-3, CRL-4, conjugate 17, conjugate 18, conjugate 19, conjugate 22,and conjugate 23.

A. Target Ligands

The targeting ligands B of the conjugates are benzazepines where abenzene ring is fused to an azepine ring. An example of a targetingligand that is used in the conjugates of the invention is thebenzodiazepine Z-360

and analogs, derivatives, salts and esters thereof.

The targeting ligands B also encompass the benzazepines of Formula I

wherein:the bond between X and Y is a single bond or a double bond;R¹=H, CH₃ or heterocycle with R²;R²=heterocycle with R¹, CH₃, CH₂CON(Et)₂, CH₂COC(CH₃)₃, CH₂CONHC(CH₃)₃,

X=CR³ or NR⁴;

R³=phenyl, cyclohexyl, CH₃, CH₂-CH₃,

R⁴=phenyl, cyclohexyl,

Y=CH₂, N (dashed=double bond), C═O;z=

R⁵=absent, phenyl, Cl, CO₂H, CH₃, OCH₃, NHCH₃, F, SCH₂CO₂H, SCH₂CO₂Et,

and analogs, derivatives, salts and esters thereof.

B. Active Moieties

The invention takes advantage of the excellent properties of theconjugates. Due to their ability to carry a “payload”, the targetingligands can be labeled and still bind to cells that express one or bothof CCK2R and CCK2i4svR. Upon binding of the receptors, the conjugatescan be detected due to the properties of an imaging agent, or exertcytotoxic effects due to the properties of a therapeutic agent. Theactive moieties D of the conjugates of the present invention are thustherapeutic agents and imaging agents. The only limitation on suitabletherapeutic agents and imaging agents is the requirement that they havea position on the molecule to which can be conjugated the linker L, orthat they can be derivatized to possess such a position without losingthe activity of the active moiety or compromising the ability of thetargeting ligand to bind to its receptor with high affinity.

Therapeutic Agents

The therapeutic agents described herein function through any of a largenumber of mechanisms of action. Generally, therapeutic agents disruptcellular mechanisms that are important for cell survival and/or cellproliferation and/or cause apoptosis. The therapeutic agents can be anycompound known in the art which is cytotoxic, enhances tumorpermeability, inhibits tumor cell proliferation, promotes apoptosis,decreases anti-apoptotic activity in target cells, is used to treatdiseases caused by infectious agents, enhances an endogenous immuneresponse directed to the pathogenic cells, or is useful for treating adisease state caused by any type of pathogenic cell.

Therapeutic agents suitable for use in accordance with this inventioninclude adrenocorticoids and corticosteroids, alkylating agents,antiandrogens, antiestrogens, androgens, aclamycin and aclamycinderivatives, estrogens, antimetabolites such as cytosine arabinoside,purine analogs, pyrimidine analogs, and methotrexate, busulfan,carboplatin, chlorambucil, cisplatin and other platinum compounds,taxanes, such as tamoxiphen, taxol, paclitaxel, paclitaxel derivatives,Taxotere®, and the like, maytansines and analogs and derivativesthereof, cyclophosphamide, daunomycin, doxorubicin, rhizoxin, T2 toxin,plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycins,discodermolides, microtubule inhibitors, epothilones, tubulysin (e.g.,tubulysin B hydrazide), cyclopropyl benz[e]indolone, seca-cyclopropylbenz[e]indolone, 0-Ac-seca-cyclopropyl benz[e]indolone, bleomycin andany other antibiotic, nitrogen mustards, nitrosureas, vincristine,vinblastine, and analogs and derivative thereof such as desacetylvinblastine monohydrazide, colchicine, colchicine derivatives,allocolchicine, thiocolchicine, trityl cysteine, Halicondrin B,dolastatins such as dolastatin 10, amanitins such as a-amanitin,camptothecin, irinotecan, and other camptothecin derivatives thereof,geldanamycin and geldanamycin derivatives, estramustine, nocodazole,MAP4, colcemid, inflammatory and proinflammatory agents, peptide andpeptidomimetic signal transduction inhibitors, and any otherart-recognized drug or toxin. Other drugs that can be used in accordancewith the invention include penicillins, dinitrophenol, fluorescein, CpGoligonucleotides, staurosporine and other kinase inhibitors, Sutent,resiquimod and other Toll-like receptor agonists, cephalosporins,vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol,aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and anyother art-recognized antimicrobial compound. The only limitation onsuitable therapeutic agents is the requirement that they have a positionon the molecule that can be conjugated to the linker L, or that they canbe derivatized to possess such a position. Illustrative therapeuticagents are described in U.S. patent application publication nos. US20050002942, US 20010031252, and US 20030086900, the disclosures of eachof which are incorporated herein by reference in their entireties.

Specific sub-groups of therapeutic agents include, but are not limitedto, radio-therapeutic agents, immunotherapeutic agents, photodynamictherapy agents and chemotherapeutic agents. The skilled artisan willunderstand that there is a wide variety of radio-therapeutics that willbe suitable for use in the conjugates of the present invention. Suitableexamples include, but are not limited to, ⁹⁰Y, ¹³¹I, ¹⁷⁷Lu, ⁶⁷Cu, ¹¹¹In,¹⁸⁶Re, ²¹¹At, and ²²³Ra.

The skilled artisan will also understand that there is a wide variety ofchemotherapeutics that will be suitable for use in the conjugates of thepresent invention. Suitable examples include, but are not limited to,tubulysin B hydrazide and desacetyl vinblastine monohydrazide,calicheamycin, auristatin, maytansinoids and any other cytotoxic agentwith IC₅₀ value below 10 nM.

It should also be appreciated that the ligand can be used to target ananomedicines or nanoparticle, including but not limited to a liposome,a lipoplex, a polyplex, a dendrimer, a polymer, a nanoparticle, or avirus. It should further be recognized that the aforementioned particlesmight serve as carriers for DNA, RNA, siRNA, peptides, proteins, andother biologics.

Imaging Agents

Imaging agents suitable for use in the conjugates of the inventioninclude, but are not limited to, radio-imaging agents, optical imagingagents, PET imaging agents, MRI contrast agents, CT contrast agents, andFRET imaging agents, and other agents that may be used to detect orvisualize a tumor, cancer or transformed cell, whether in vitro, in vivoand ex vivo. Illustrative imaging agents are described in U.S. patentapplication publication no. US 20040033195 and international patentapplication publication no. WO 03/097647, the disclosures of each ofwhich are incorporated herein by reference in their entireties.

Applications for conjugates comprising radio-imaging agents include andmay not be limited to diagnosis of disease and or locating metastaticdisease, detecting disease recurrence following surgery, monitoringresponse to therapy, development of a radio-therapeutic conjugate andselecting patients for subsequent CCK2R targeted therapy. Radio-imagingagents include radioactive isotopes, such as a radioactive isotope of ametal, coordinated to a chelating group. Illustrative radioactive metalisotopes include technetium, rhenium, gallium, gadolinium, indium,copper, and the like, including isotopes ¹¹¹In, ⁹⁹mTc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga,and the like, or they may include radionuclides that are effective inradiotherapy. Additional illustrative examples of radionuclide imagingagents are described in U.S. Pat. No. 7,128,893, the disclosure of whichis incorporated herein by reference in its entirety.

Illustratively, the following chelating groups are described that can beused with the radio-imaging agents:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L, and n is an integer from 1 to about 5.

Additional illustrative chelating groups are tripeptide ortetrapeptides, including but not limited to tripeptides having theformula:

wherein R is independently selected in each instance from H, alkyl,heteroalkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, and the like, each of which isoptionally substituted. It is to be understood that one R includes aheteroatom, such as nitro, oxygen, or sulfur, and is the point ofattachment of linker L.

Applications for conjugates comprising optical imaging agents includelocating and resecting large tumor masses, delineation of normal andmalignant tissue, intraoperative detection of sentinel lymph nodes, andfluorescent probe for minimally invasive laparoscopic procedures as analternative to second look surgery. The skilled artisan will alsounderstand that there is a wide variety of optical imaging agents thatwill be suitable for use in the conjugates of the present invention. Theonly limitations on suitable optical imaging agents is the requirementthat they have a position on the molecule to which can be conjugated thelinker L, or that they can be derivatized to possess such a position.Examples include, but are not limited to, Oregon Green fluorescentagents, including but not limited to Oregon Green 488, Oregon Green 514,and the like, AlexaFluor fluorescent agents, including but not limitedto AlexaFluor 488, AlexaFluor 647, and the like, fluorescein, andrelated analogs, BODIPY fluorescent agents, including but not limited toBODIPY F1, BODIPY 505, 50456, and the like, rhodamine fluorescentagents, including but not limited to tetramethylrhodamine, and the like,near infra-red fluorescent agents, including but not limited to DyLight680, DyLight 800, 800CW, LS288, 50456, indocyanine green and the like,Texas Red, phycoerythrin, and others. Illustrative optical imagingagents are shown in the following general structure:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; Y is ORa, NRa₂, or NRa₃₊; and Y′ is O, NRa, or NRa₂₊; whereeach R is independently selected in each instance from H, fluoro,sulfonic acid, sulfonate, and salts thereof, and the like; and Ra ishydrogen or alkyl.

According to another aspect, illustrative optical imaging agents areshown in the following general structure:

where X is oxygen, nitrogen, or sulfur, and where X is attached tolinker L; and each R is independently selected in each instance from H,alkyl, heteroalkyl, and the like; and n is an integer from 0 to about 4.

The skilled artisan will also understand that there is a wide variety ofPET imaging agents and FRET imaging agents that will be suitable for usein the conjugates of the present invention. The only limitations onsuitable PET and FRET imaging agents is the requirement that they have aposition on the molecule to which can be conjugated the linker L, orthat they can be derivatized to possess such a position. Examples of PETimaging agents include, but are not limited to, ¹⁸F, ¹¹C, ⁶⁴CU, ⁶⁵Cu,and the like. Examples of FRET imaging agents include, but are notlimited to, ⁶⁴eu, ⁶⁵eu, and the like. It appreciated that in the case of¹⁸F and ¹¹C, the imaging isotope may be present on any part of thelinker, or alternatively may be present on a structure attached to thelinker. For example in the case of ¹⁸F, fluoroaryl groups, such asfluorophenyl, difluorophenyl, fluoronitrophenyl, and the like aredescribed. For example in the case of ¹¹C, alkyl and alkyl aryl aredescribed.

Exemplary optical imaging agents include, but are not limited to,fluorescein (FITC), rhodamine, LS288, 50456, IR800CW, or another nearinfrared dye.

C. Linkers

The targeting ligand B, or analog or derivative thereof, is covalentlyattached to the polyvalent linker L, and the active moiety D, or analogor derivative thereof, is also covalently attached to the polyvalentlinker L. Exemplary linkers include, but are not limited to, ahydrophilic linkers comprised of charged or polar amino acids, sugars orsugar-containing oligomers, and hydrophilic polymers such aspolyethylene glycol.

In a first embodiment, the linker L is L1, L2 or L3

L1: HN-Glu-Arg-Asp-CO

L2: HN-Glu-PS-Glu-PS-CO

L3: HN-Octanoyl-Glu-PS-Glu-PS-CO

wherein PS is the following formula

The linkers used in the production of the conjugates may also compriseone or more spacer linkers and/or one or more releasable linkers, andcombinations thereof, in any order. It is appreciated that spacerlinkers may included when predetermined lengths are selected forseparating targeting ligand from the active moiety. It is alsoappreciated that in certain configurations, releasable linkers may beincluded. For example, as described herein in one embodiment, theconjugates may be used to deliver therapeutic agents for treating canceror other diseases involving pathogenic cells. In such embodiments, it isappreciated that once delivered, the therapeutic agent is desirablyreleased from the conjugate.

In one variation, releasable linkers, and optional spacer linkers arecovalently bonded to each other to form the linker. In anothervariation, a releasable linker is directly attached to the activemoiety, or analog or derivative thereof. In another variation, areleasable linker is directly attached to the targeting ligand. Inanother variation, either or both the targeting ligand and the activemoiety, or analog or derivative thereof, is attached to a releasablelinker through one or more spacer linkers. In another variation, each ofthe targeting ligand and the active moiety, or analog or derivativethereof, is attached to a releasable linker, each of which may bedirectly attached to each other, or covalently attached through one ormore spacer linkers.

From the foregoing, it should be appreciated that the arrangement of thetargeting ligand, and the active moieties, or analogs or derivativesthereof, and the various releasable and optional spacer linkers may bevaried widely. In one aspect, the targeting ligand and the activemoiety, and the various releasable and optional spacer linkers areattached to each other through heteroatoms, such as nitrogen, oxygen,sulfur, phosphorus, silicon, and the like. In variations, theheteroatoms, excluding oxygen, may be in various states of oxidation,such as N(OH), S(O), S(O)₂, P(O), P(O)₂, P(O)₃, and the like. In othervariation, the heteroatoms may be grouped to form divalent radicals,such as for example hydroxylamines, hydrazines, hydrazones, sulfonates,phosphinates, phosphonates, and the like, including radicals of theformulae —(NHR₁NHR₂)—, —SO—, —(SO₂)—, and —N(R₃)O—, wherein R₁, R₂, andR₃ are each independently selected from hydrogen, alkyl, aryl,arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl,substituted heteroaryl, and alkoxyalkyl. In another variation, more thanone targeting ligand is attached to the polyvalent linker. In anothervariation, more than one active moiety is attached to the polyvalentlinker. In another variation, more than one targeting ligand and morethan one active moiety is attached to the polyvalent linker.

It is appreciated that the arrangement and/or orientation of the varioushydrophilic linkers may be in a linear or branched fashion, or both. Forexample, the hydrophilic linkers may form the backbone of the linkerforming the conjugate between the targeting ligand and the activemoiety. Alternatively, the hydrophilic portion of the linker may bependant to or attached to the backbone of the chain of atoms connectingthe targeting ligand to the active moiety. In this latter arrangement,the hydrophilic portion may be proximal or distal to the backbone chainof atoms.

In another embodiment, the linker is more or less linear, and thehydrophilic groups are arranged largely in a series to form a chain-likelinker in the conjugate. Said another way, the hydrophilic groups formsome or all of the backbone of the linker in this linear embodiment.

In another embodiment, the linker is branched with hydrophilic groups.In this branched embodiment, the hydrophilic groups may be proximal tothe backbone or distal to the backbone. In each of these arrangements,the linker is more spherical or cylindrical in shape.

In one variation, the linker is shaped like a bottle-brush. In oneaspect, the backbone of the linker is formed by a linear series ofamides, and the hydrophilic portion of the linker is formed by aparallel arrangement of branching side chains, such as by connectingmonosaccharides, sulfonates, and the like, and derivatives and analogsthereof.

It is understood that the linker may be neutral or ionizable undercertain conditions, such as physiological conditions encountered invivo. For ionizable linkers, under the selected conditions, the linkermay deprotonate to form a negative ion, or alternatively becomeprotonated to form a positive ion. It is appreciated that more than onedeprotonation or protonation event may occur. In addition, it isunderstood that the same linker may deprotonate and protonate to forminner salts or zwitterionic compounds.

In another embodiment, the hydrophilic spacer linkers are neutral, i.e.under physiological conditions, the linkers do not significantlyprotonate nor deprotonate. In another embodiment, the hydrophilic spacerlinkers may be protonated to carry one or more positive charges. It isunderstood that the protonation capability is condition dependent. Inone aspect, the conditions are physiological conditions, and the linkeris protonated in vivo. In another embodiment, the spacers include bothregions that are neutral and regions that may be protonated to carry oneor more positive charges. In another embodiment, the spacers includeboth regions that may be deprotonated to carry one or more negativecharges and regions that may be protonated to carry one or more positivecharges. It is understood that in this latter embodiment thatzwitterions or inner salts may be formed.

In one aspect, the regions of the linkers that may be deprotonated tocarry a negative charge include carboxylic acids, such as aspartic acid,glutamic acid, and longer chain carboxylic acid groups, and sulfuricacid esters, such as alkyl esters of sulfuric acid. In another aspect,the regions of the linkers that may be protonated to carry a positivecharge include amino groups, such as polyaminoalkylenes includingethylene diamines, propylene diamines, butylene diamines and the like,and/or heterocycles including pyrollidines, piperidines, piperazines,and other amino groups, each of which is optionally substituted. Inanother embodiment, the regions of the linkers that are neutral includepoly hydroxyl groups, such as sugars, carbohydrates, saccharides,inositols, and the like, and/or polyether groups, such aspolyoxyalkylene groups including polyoxyethylene, polyoxypropylene, andthe like.

In one embodiment, the hydrophilic spacer linkers described hereininclude are formed primarily from carbon, hydrogen, and oxygen, and havea carbon/oxygen ratio of about 3:1 or less, or of about 2:1 or less. Inone aspect, the hydrophilic linkers described herein include a pluralityof ether functional groups. In another aspect, the hydrophilic linkersdescribed herein include a plurality of hydroxyl functional groups.Illustrative fragments that may be used to form such linkers includepolyhydroxyl compounds such as carbohydrates, polyether compounds suchas polyethylene glycol units, and acid groups such as carboxyl and alkylsulfuric acids. In one variation, oligoamide spacers, and the like mayalso be included in the linker.

In one illustrative embodiment, conjugates of the present inventioninclude linkers having predetermined length and diameter dimensions. Inone aspect, linkers are described herein that satisfy one or moreminimum length requirements, or a length requirement falling within apredetermined range. In another aspect, satisfaction of a minimum lengthrequirement may be understood to be determined by computer modeling ofthe extended conformations of linkers. In another aspect, satisfactionof a minimum length requirement may be understood to be determined byhaving a certain number of atoms, whether or not substituted, forming abackbone chain of atoms connecting the target ligand with the activemoiety. In another embodiment, the backbone chain of atoms is cyclizedwith another divalent fragment. In another aspect, linkers are describedherein that satisfy one or more maximum or minimum diameterrequirements. In another aspect, satisfaction of a maximum or minimumdiameter requirement may be understood to be determined by computermodeling of various conformations of linkers modeled as thespace-filling, CPK, or like configurations. In another aspect,satisfaction of a maximum or minimum diameter requirement may beunderstood to be apply to one or more selected portions of the linker,for example the portion of the linker proximal to the targeting ligand,or the portion of the linker proximal to the active moiety, and thelike. In another aspect, linkers are described herein that satisfy oneor more chemical composition requirements, such as linkers that includeone or more polar groups that may positively interact with the one ormore side chains found in the CCK2R receptor. In one variation, linkersare described herein that satisfy one or more chemical compositionrequirements, such as linkers that include one or more non-polar groupsthat may positively interact with the CCK2R receptor.

In another embodiment, linkers are described that include at least onereleasable linker. In one variation, linkers are described that includeat least two releasable linkers. In another variation, linkers aredescribed that include at least one self-immolative linker. In anothervariation, linkers are described that include at least one releasablelinker that is not a disulfide. In another embodiment, linkers aredescribed that do not include a releasable linker.

It is appreciated that releasable linkers may be used when the activemoiety to be delivered is advantageously liberated from the targetingligand-linker conjugate so that free active moiety will have the same ornearly the same effect at the target as it would when administeredwithout the targeting provided by the conjugates described herein. Inanother embodiment, the linker is a non-releasable linker. It isappreciated that non-releasable linkers may be used when the activemoiety is advantageously retained by the targeting ligand-linkerconjugate, such as in imaging, diagnostic, uses of the conjugatesdescribed herein.

It is to be understood that the choice of a releasable linker or anon-releasable linker may be made independently for each application orconfiguration of the conjugates, without limiting the inventiondescribed herein.

It is to be further understood that the linkers described hereincomprise various atoms, chains of atoms, functional groups, andcombinations of functional groups. Where appropriate in the presentdisclosure, the linker may be referred to by the presence of spacerlinkers, releasable linkers, and heteroatoms. However, such referencesare not to be construed as limiting the definition of the linkersdescribed herein.

The linker comprising spacer and/or releasable linkers (i.e., cleavablelinkers) can be any biocompatible linker. The releasable or cleavablelinker can be, for example, a linker susceptible to cleavage under thereducing or oxidizing conditions present in or on cells, a pH-sensitivelinker that may be an acid-labile or base-labile linker, or a linkerthat is cleavable by biochemical or metabolic processes, such as anenzyme-labile linker. According to at least one embodiment, the spacerand/or releasable linker comprises about 1 to about 30 atoms, or about 2to about 20 atoms. Lower molecular weight linkers (i.e., those having anapproximate molecular weight of about 30 to about 300) are alsodescribed. Precursors to such linkers may be selected to have eithernucleophilic or electrophilic functional groups, or both, optionally ina protected form with a readily cleavable protecting group to facilitatetheir use in synthesis of the intermediate species.

The term “releasable linker” as used herein refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, redox-labile, orenzyme-labile bond). The cleavable bond or bonds may be present in theinterior of a cleavable linker and/or at one or both ends of a cleavablelinker. It should be appreciated that such physiological conditionsresulting in bond breaking include standard chemical hydrolysisreactions that occur, for example, at physiological pH, or as a resultof compartmentalization into a cellular organelle such as an endosomehaving a lower pH than cytosolic pH. Illustratively, the bivalentlinkers described herein may undergo cleavage under other physiologicalor metabolic conditions, such as by the action of a glutathione mediatedmechanism. It is appreciated that the lability of the cleavable bond maybe adjusted by including functional groups or fragments within thebivalent linker that are able to assist or facilitate such bondbreakage, also termed anchimeric assistance. The lability of thecleavable bond can also be adjusted by, for example, substitutionalchanges at or near the cleavable bond, such as including alpha branchingadjacent to a cleavable disulfide bond, increasing the hydrophobicity ofsubstituents on silicon in a moiety having a silicon-oxygen bond thatmay be hydrolyzed, homologating alkoxy groups that form part of a ketalor acetal that may be hydrolyzed, and the like. In addition, it isappreciated that additional functional groups or fragments may beincluded within the bivalent linker that are able to assist orfacilitate additional fragmentation of the conjugates after bondbreaking of the releasable linker.

In another embodiment, the linker includes radicals that form one ormore spacer linkers and/or releasable linkers that are taken together toform the linkers described herein having certain length, diameter,and/or functional group requirements.

Another illustrative embodiment of the linkers described herein, includereleasable linkers that cleave under the conditions described herein bya chemical mechanism involving beta elimination. In one aspect, suchreleasable linkers include beta-thio, betahydroxy, and beta-aminosubstituted carboxylic acids and derivatives thereof, such as esters,amides, carbonates, carbamates, and ureas. In another aspect, suchreleasable linkers include 2- and 4-thioarylesters, carbamates, andcarbonates.

It is to be understood that releasable linkers may also be referred toby the functional groups they contain, illustratively such as disulfidegroups, ketal groups, and the like, as described herein. Accordingly, itis understood that a cleavable bond can connect two adjacent atomswithin the releasable linker and/or connect other linkers, or thetargeting ligand, or the active moiety, as described herein, at eitheror both ends of the releasable linker. In the case where a cleavablebond connects two adjacent atoms within the releasable linker, followingbreakage of the bond, the releasable linker is broken into two or morefragments. Alternatively, in the case where a cleavable bond is betweenthe releasable linker and another moiety, such as an additionalheteroatom, a spacer linker, another releasable linker, the activemoiety, or analog or derivative thereof, or the targeting ligand, oranalog or derivative thereof, following breakage of the bond, thereleasable linker is separated from the other moiety.

In another embodiment, the releasable and spacer linkers may be arrangedin such a way that subsequent to the cleavage of a bond in the bivalentlinker, released functional groups anchimerically assist the breakage orcleavage of additional bonds, as described above.

An illustrative embodiment of such a bivalent linker or portion thereofincludes compounds having the formula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and the symbol (*) indicates points of attachment for additionalspacer or releasable linkers, or heteroatoms, forming the bivalentlinker, or alternatively for attachment of the active moiety, or analogor derivative thereof, or the targeting ligand, or analog or derivativethereof. It is appreciated that other substituents may be present on thearyl ring, the benzyl carbon, the alkanoic acid, or the methylenebridge, including but not limited to hydroxy, alkyl, alkoxy, alkylthio,halo, and the like. Assisted cleavage may include mechanisms involvingbenzylium intermediates, benzyne intermediates, lactone cyclization,oxonium intermediates, beta-elimination, and the like. It is furtherappreciated that, in addition to fragmentation subsequent to cleavage ofthe releasable linker, the initial cleavage of the releasable linker maybe facilitated by an anchimerically-assisted mechanism.

In this embodiment, the hydroxyalkanoic acid, which may cyclize,facilitates cleavage of the methylene bridge, by for example an oxoniumion, and facilitates bond cleavage or subsequent fragmentation afterbond cleavage of the releasable linker. Alternatively, acid catalyzedoxonium ion-assisted cleavage of the methylene bridge may begin acascade of fragmentation of this illustrative bivalent linker, orfragment thereof. Alternatively, acid-catalyzed hydrolysis of thecarbamate may facilitate the beta elimination of the hydroxyalkanoicacid, which may cyclize, and facilitate cleavage of methylene bridge, byfor example an oxonium ion. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation.

Illustrative mechanisms for cleavage of the bivalent linkers describedherein include the following 1,4 and 1,6 fragmentation mechanisms

where X is an exogenous or endogenous nucleophile, glutathione, orbioreducing agent, and the like, and either of Z or Z′ is a targetingligand, or an active moiety, or either of Z or Z′ is a targeting ligand,or an active moiety connected through other portions of the bivalentlinker. It is to be understood that although the above fragmentationmechanisms are depicted as concerted mechanisms, any number of discretesteps may take place to effect the ultimate fragmentation of thebivalent linker to the final products shown. For example, it isappreciated that the bond cleavage may also occur by acid catalyzedelimination of the carbamate moiety, which may be anchimericallyassisted by the stabilization provided by either the aryl group of thebeta sulfur or disulfide illustrated in the above examples. In thosevariations of this embodiment, the releasable linker is the carbamatemoiety. Alternatively, the fragmentation may be initiated by anucleophilic attack on the disulfide group, causing cleavage to form athiolate. The thiolate may intermolecularly displace a carbonic acid orcarbamic acid moiety and form the corresponding thiacyclopropane. In thecase of the benzyl-containing bivalent linkers, following anillustrative breaking of the disulfide bond, the resulting phenylthiolate may further fragment to release a carbonic acid or carbamicacid moiety by forming a resonance stabilized intermediate. In any ofthese cases, the releaseable nature of the illustrative bivalent linkersdescribed herein may be realized by whatever mechanism may be relevantto the chemical, metabolic, physiological, or biological conditionspresent.

Other illustrative mechanisms for bond cleavage of the releasable linkerinclude oxonium-assisted cleavage as follows:

where Z is the targeting ligand, or analog or derivative thereof, or theactive moiety, or analog or derivative thereof, or each is a targetingligand or active moiety in conjunction with other components of thepolyvalent linker, such as an active moiety or targeting ligandincluding one or more spacer linkers and/or other releasable linkers. Inthis embodiment, acid-catalyzed elimination of the carbamate leads tothe release of CO₂ and the nitrogen-containing moiety attached to Z, andthe formation of a benzyl cation, which may be trapped by water, or anyother Lewis base.

According to at least one embodiment, the releasable linker includes adisulfide.

In another embodiment, the releasable linker may be a divalent radicalcomprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl,carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, oralkylenesulfonylaziridin-1-yl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below.

Additional illustrative releasable linkers include methylene,1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsil yl),alkylene(diarylsil yl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy,oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl,aminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of the releasablelinkers is optionally substituted with a substituent X², as definedbelow.

In the preceding embodiment, the releasable linker may include oxygen,and the releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the oxygen to form an acetal or ketal.Alternatively, the releasable linker may include oxygen, and thereleasable linker can be methylene, wherein the methylene is substitutedwith an optionally-substituted aryl, and the releasable linker is bondedto the oxygen to form an acetal or ketal. Further, the releasable linkermay include oxygen, and the releasable linker can be sulfonylalkyl, andthe releasable linker is bonded to the oxygen to form an alkylsulfonate.

In another embodiment of the above releasable linker embodiment, thereleasable linker may include nitrogen, and the releasable linkers canbe aminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, andcarbonylcycloalkylideniminyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the nitrogen to form an hydrazone. In analternate configuration, the hydrazone may be acylated with a carboxylicacid derivative, an orthoformate derivative, or a carbamoyl derivativeto form various acylhydrazone releasable linkers.

Alternatively, the releasable linker may include oxygen, and thereleasable linkers can be alkylene(dialkylsilyl),alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl,(alkylarylsilyl)aryl, and (diarylsilyl)aryl, wherein each of thereleasable linkers is optionally substituted with a substituent X², asdefined below, and the releasable linker is bonded to the oxygen to forma silanol.

In the above releasable linker embodiment, the active moiety can includea nitrogen atom, the releasable linker may include nitrogen, and thereleasable linkers can be carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, and thereleasable linker can be bonded to the heteroatom nitrogen to form anamide, and also bonded to the active moiety nitrogen to form an amide.

In the above releasable linker embodiment, the active moiety can includean oxygen atom, the releasable linker may include nitrogen, and thereleasable linkers can be carbonylarylcarbonyl,carbonyll(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl, andthe releasable linker can form an amide, and also bonded to the activemoiety oxygen to form an ester.

The substituents X² can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴ carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides.

In this embodiment the releasable linker can include nitrogen, and thesubstituent X² and the releasable linker can form a heterocycle. Theheterocycles can be pyrrolidines, piperidines, oxazolidines,isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones,piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones,isothiazolidinones, and succinimides.

In a specific embodiment, the linkers for use in the conjugates of thepresent invention include the following:

where n is an integer selected from 1 to about 4; R^(a) and R^(b) areeach independently selected from the group consisting of hydrogen andalkyl, including lower alkyl such as C₁-C₄ alkyl that are optionallybranched; or R^(a) and R^(b) are taken together with the attached carbonatom to form a carbocyclic ring; R is an optionally substituted alkylgroup, an optionally substituted acyl group, or a suitably selectednitrogen protecting group; and (*) indicates interchangeable points ofattachment for the targeting ligand and the active moiety.

In a further specific embodiment, the linkers for use in the conjugatesof the present invention include the following:

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicatesinterchangeable points of attachment for the targeting ligand and theactive moiety.

In an additional specific embodiment, the linkers for use in theconjugates of the present invention include the following:

where m is an integer selected from 1 to about 4; R is an optionallysubstituted alkyl group, an optionally substituted acyl group, or asuitably selected nitrogen protecting group; and (*) indicatesinterchangeable points of attachment for the targeting ligand and theactive moiety.

In a further additional specific embodiment, the linkers for use in theconjugates of the present invention include the following:

wherein n and m are each independently selected integers from 1 to about4; R^(a) and R^(b) are each independently selected from the groupconsisting of hydrogen and alkyl, including lower alkyl such as C₁-C₄alkyl that are optionally branched; or R^(a) and R^(b) are takentogether with the attached carbon atom to form a carbocyclic ring; andX¹ and X² are each independently selected leaving groups that may benucleophilically displaced by a targeting ligand, an active moiety,another bivalent linker, or another part of the conjugate.

Additional linkers and means for preparing the linkers of the precedingfour embodiments are provided in US 2009/0203889 and WO 2006/012527, thedisclosures of both of which are incorporated herein by reference intheir entireties.

In another embodiment, the linker includes one or more spacer linkers.Such spacer linkers can be 1-alkylenesuccinimid-3-yl, optionallysubstituted with a substituent X¹, as defined below, and the releasablelinkers can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein each ofthe releasable linkers is optionally substituted with a substituent X²,as defined above, and wherein the spacer linker and the releasablelinker are each bonded to the spacer linker to form asuccinimid-1-ylalkyl acetal or ketal.

The spacer linkers can be carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the spacerlinkers is optionally substituted with a substituent X¹ as definedbelow. In this embodiment, the spacer linker may include an additionalnitrogen, and the spacer linkers can be alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹ as defined below, and thespacer linker is bonded to the nitrogen to form an amide. Alternatively,the spacer linker may include an additional sulfur, and the spacerlinkers can be alkylene and cycloalkylene, wherein each of the spacerlinkers is optionally substituted with carboxy, and the spacer linker isbonded to the sulfur to form a thiol. In another embodiment, the spacerlinker can include sulfur, and the spacer linkers can be1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and thespacer linker is bonded to the sulfur to form a succinimid-3-ylthiol.

In an alternative to the above-described embodiments, the spacer linkercan include nitrogen, and the releasable linker can be a divalentradical comprising alkyleneaziridin-1-yl, carbonylalkylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, or sulfonylalkylaziridin-1-yl, wherein eachof the releasable linkers is optionally substituted with a substituentX², as defined above. In this alternative embodiment, the spacer linkerscan be carbonyl, thionocarbonyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹ as defined below, andwherein the spacer linker is bonded to the releasable linker to form anaziridine amide.

The substituents X¹ can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the spacer linker caninclude nitrogen, and the substituent X¹ and the spacer linker to whichthey are bound to form a heterocycle.

Additional illustrative spacer linkers includealkylene-amino-alkylenecarbonyl,alkylene-thio-(carbonylalkylsuccinimid-3-yl), and the like, as furtherillustrated by the following formulae:

where the integers x and y are 1, 2, 3, 4, or 5.

In another embodiment, linkers that include hydrophilic regions are alsodescribed. In one aspect, the hydrophilic region of the linker formspart or all of a spacer linker included in the conjugates describedherein. Illustrative hydrophilic spacer linkers are described in WO2008/002993, published Dec. 31, 2008, the disclosure of which isincorporated herein by reference.

The term “cycloalkyl” as used herein includes molecular fragments orradicals comprising a bivalent chain of carbon atoms, a portion of whichforms a ring. It is to be understood that the term cycloalkyl as usedherein includes fragments and radicals attached at either ring atoms ornon-ring atoms, such as, such as cyclopropyl, cyclohexyl,3-ethylcyclopent-1-yl, cyclopropylethyl, cyclohexylmethyl, and the like.

The term “cycloalkylene” as used herein includes molecular fragments orradicals comprising a bivalent chain of carbon atoms, a portion of whichforms a ring. It is to be understood that the term cycloalkyl as usedherein includes fragments and radicals attached at either ring atoms ornon-ring atoms, such as cycloprop-1,1-diyl, cycloprop-1,2-diyl,cyclohex-1,4-diyl, 3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl,and the like.

The terms “heteroalkyl” and “heteroalkylene” as used herein includesmolecular fragments or radicals comprising monovalent and divalent,respectively, groups that are formed from a linear or branched chain ofcarbon atoms and heteroatoms, wherein the heteroatoms are selected fromnitrogen, oxygen, and sulfur, such as alkoxyalkyl, alkyleneoxyalkyl,aminoalkyl, alkylaminoalkyl, alkyleneaminoalkyl, alkylthioalkyl,alkylenethioalkyl, alkoxyalkylaminoalkyl, alkylaminoalkoxyalkyl,alkyleneoxyalkylaminoalkyl, and the like.

The term “heterocyclyl” as used herein includes molecular fragments orradicals comprising a monovalent chain of carbon atoms and heteroatoms,wherein the heteroatoms are selected from nitrogen, oxygen, and sulfur,a portion of which, including at least one heteroatom, form a ring, suchas aziridine, pyrrolidine, oxazolidine, 3-methoxypyrrolidine,3-methylpiperazine, and the like. Accordingly, as used herein,heterocyclyl includes alkylheterocyclyl, heteroalkylheterocyclyl,heterocyclylalkyl, heterocyclylheteroalkyl, and the like. It is to beunderstood that the term heterocyclyl as used herein includes fragmentsand radicals attached at either ring atoms or non-ring atoms, such astetrahydrofuran-2-yl, piperidin-1-yl, piperidin-4-yl, piperazin-1-yl,morpholin-1-yl, tetrahydrofuran-2-ylmethyl, piperidin-1-ylethyl,piperidin-4-ylmethyl, piperazin-1-ylpropyl, morpholin-1-ylethyl, and thelike.

The term “aryl” as used herein includes molecular fragments or radicalscomprising an aromatic mono or polycyclic ring of carbon atoms, such asphenyl, naphthyl, and the like.

The term “heteroaryl” as used herein includes molecular fragments orradicals comprising an aromatic mono or polycyclic ring of carbon atomsand at least one heteroatom selected from nitrogen, oxygen, and sulfur,such as pyridinyl, pyrimidinyl, indolyl, benzoxazolyl, and the like.

The term “substituted aryl” or “substituted heteroaryl” as used hereinincludes molecular fragments or radicals comprising aryl or heteroarylsubstituted with one or more substituents, such as alkyl, heteroalkyl,halo, hydroxy, amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl,aminosulfonyl, carboxylate, alkoxycarbonyl, aminocarbonyl, cyano, nitro,and the like. It is to be understood that the alkyl groups in suchsubstituents may be optionally substituted with halo.

The term “iminoalkylidenyl” as used herein includes molecular fragmentsor radicals comprising a divalent radical containing alkylene as definedherein and a nitrogen atom, where the terminal carbon of the alkylene isdouble-bonded to the nitrogen atom, such as the formulae —(CH)═N—,—(CH₂)2(CH)═N—, —CH₂C(Me)═N—, and the like.

The term “amino acid” as used herein includes molecular fragments orradicals comprising an aminoalkylcarboxylate, where the alkyl radical isoptionally substituted with alkyl, hydroxy alkyl, sulfhydrylalkyl,aminoalkyl, carboxyalkyl, and the like, including groups correspondingto the naturally occurring amino acids, such as serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

For example, and according to at least one embodiment, amino acid is adivalent radical having the general formula:

—N(R)—(CR′R″)q—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornitine, threonine, and thelike. In one variation, the amino acid may be selected fromphenylalanine, tyrosine, and the like, derivatives thereof, andsubstituted variants thereof.

The terms “arylalkyl” and “heteroarylalkyl” as used herein includesmolecular fragments or radicals comprising aryl and heteroaryl,respectively, as defined herein substituted with a linear or branchedalkylene group, such as benzyl, phenethyl, α-methylbenzyl, picolinyl,pyrimidinylethyl, and the like.

It is to be understood that the above-described terms can be combined togenerate chemically-relevant groups, such as “haloalkoxyalkyl” referringto for example trifluoromethyloxyethyl,1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.

The term “amino acid derivative” as used herein refers generally toaminoalkylcarboxylate, where the amino radical or the carboxylateradical are each optionally substituted with alkyl, carboxylalkyl,alkylamino, and the like, or optionally protected; and the interveningdivalent alkyl fragment is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like,including groups corresponding to the side chains found in naturallyoccurring amino acids, such as are found in serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

The term “peptide” as used herein includes molecular fragments orradicals comprising a series of amino acids and amino acid analogs andderivatives covalently linked one to the other by amide bonds.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is optionallysubstituted with alkyl or substituted aryl.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms anacylaziridine with the active moiety, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form 1-alkoxycycloalkylenoxy.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to formalkyleneaminocarbonyl(dicarboxylarylene)carboxylate.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to formdithioalkylcarbonylhydrazide, where the hydrazide forms a hydrazone withthe active moiety, or analog or derivative thereof. In anotherembodiment, the bivalent linker comprises a spacer linker and areleasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide formsan hydrazone with the active moiety, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where thedisubstituted silyl is substituted with alkyl or optionally substitutedaryl.

In another embodiment, the bivalent linker comprises a plurality ofspacer linkers selected from the group consisting of the naturallyoccurring amino acids and stereoisomers thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with theactive moiety, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate withthe active moiety, or analog or derivative thereof, and the aryl isoptionally substituted.

In another embodiment, the bivalent linker comprises a spacer linker anda releasable linker taken together to form3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylideneforms an hydrazone with the active moiety, or analog or derivativethereof, each alkyl is independently selected, and the oxyalkyloxy isoptionally substituted with alkyl or optionally substituted aryl.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonylhydrazide.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with theactive moiety, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with theactive moiety, or analog or derivative thereof, and the alkyl is ethyl.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe active moiety, or analog or derivative thereof.

In another embodiment, the bivalent linker comprises a releasablelinker, a spacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe active moiety, or analog or derivative thereof, and the alkyl isethyl.

In another embodiment, the polyvalent linker includes additional spacerlinkers and releasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkyloxymethyloxy group, illustrated by thefollowing formula

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the methyl is optionally substituted with an additionalalkyl or optionally substituted aryl group, each of which is representedby an independently selected group R. The (*) symbols indicate points ofattachment of the polyvalent linker fragment to other parts of theconjugates described herein.

In another embodiment, the polyvalent linker includes additional spacerlinkers and releasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkylcarbonyl group, illustrated by the followingformula

where n is an integer from 1 to 6, and the alkyl group is optionallysubstituted. The (*) symbols indicate points of attachment of thepolyvalent linker fragment to other parts of the conjugates describedherein. In another embodiment, the polyvalent linker includes spacerlinkers and releasable linkers connected to form a polyvalent3-thioalkylsulfonylalkyl(disubstituted silyl)oxy group, where thedisubstituted silyl is substituted with alkyl and/or optionallysubstituted aryl groups.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalentdithioalkylcarbonylhydrazide group, or a polyvalent3-thiosuccinimid-1-ylalkylcarbonylhydrazide, illustrated by thefollowing formulae

where n is an integer from 1 to 6, the alkyl group is optionallysubstituted, and the hydrazide forms an hydrazone with (B), (D), oranother part of the polyvalent linker (L). The (*) symbols indicatepoints of attachment of the polyvalent linker fragment to other parts ofthe conjugates described herein.

In another embodiment, the polyvalent linker includes spacer linkers andreleasable linkers connected to form a polyvalent3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group, illustrated bythe following formula

where each n is an independently selected integer from 1 to 6, eachalkyl group independently selected and is optionally substituted, suchas with alkyl or optionally substituted aryl, and where the alkylideneforms an hydrazone with the targeting ligand, the active moiety, oranother part of the polyvalent linker. The (*) symbols indicate pointsof attachment of the polyvalent linker fragment to other parts of theconjugates described herein.

Additional illustrative linkers are described in WO 2006/012527, thedisclosure of which is incorporated herein by reference. Additionallinkers are described in the following Table 1, where the (*) atom isthe point of attachment of additional spacer or releasable linkers, theactive moiety, and/or the targeting ligand.

TABLE 1

As suggested above, the conjugates of the invention may have 1, 2, 3, 4or 5 active moieties linked to a single targeting ligand. In particularaspects of the invention, only one active moiety will be linked to asingle targeting ligand. However, when there are two or more activemoieties linked to a single targeting ligand, the active moieties may bethe same or different. For example, where there are two active moietieslinked to a single targeting ligand, the active moieties may be twodifferent therapeutic agents, two different imaging agents, or atherapeutic agent and an imaging agent.

It is to be understood that analogs and derivatives of each of theforegoing B, L, and D are also contemplated and described herein, andthat when used herein, the terms targeting moiety (or B), linker (or L),and active moiety (or D) collectively refer to such analogs andderivatives.

III. Methods of Detection, Diagnosis and Imaging

The present invention also includes methods of using the conjugates in avariety of applications, such as in methods of detecting, diagnosing andimaging cancer. For example, in one aspect the invention is directed tomethods for detecting a tumor in a subject, comprising administering aconjugate as defined herein to a subject suspected of having a tumor anddetecting the conjugate in the subject, wherein the active moiety D ofthe conjugate is an imaging agent. In a related aspect, the inventionincludes methods for diagnosing cancer in a subject, comprisingadministering a conjugate as defined herein to a subject suspected ofhaving cancer and detecting the conjugate in the subject, wherein theactive moiety D of the conjugate is an imaging agent. In a furtherrelated aspect, the invention includes methods for imaging cancer in asubject, comprising administering a conjugate as defined herein to asubject suspected of or having cancer and detecting the conjugate in thesubject, wherein the active moiety D of the conjugate is an imagingagent.

IV. Methods of Treatment

In another aspect, the invention is directed to methods for treating asubject having cancer, comprising administering atherapeutically-effective amount of a conjugate as defined herein to asubject having cancer, wherein the active moiety D of the conjugate is atherapeutic agent. Upon binding of the receptors, the therapeutic agentcan exert an effect, either directly on the cell to which it is bound orto neighboring cells, due to the properties of the therapeutic agent.

V Pharmaceutical Formulations

The methods of the present invention using the conjugates may bepracticed in one or more of in vitro, in vivo and ex vivo applications.When used in vivo and ex vivo, the conjugates may be prepared as apharmaceutical composition comprising the conjugate and one or morepharmaceutically-acceptable carriers and/or diluents. The skilledartisan will understand that the specific elements comprising apharmaceutical composition will depend on factors that include theidentity of the targeting ligand and the active moiety in the conjugate,the identity of the cancer or tumor to be detected or treated, thelocation in the subject of the cancer or tumor, available means fordetecting the imaging agent, and the means used to administer theconjugate to the subject.

The pharmaceutical compositions of the present invention may beformulated, for example, for oral, sublingual, intranasal, intraocular,rectal, transdermal, mucosal, pulmonary, topical or parenteraladministration. Parenteral modes of administration include withoutlimitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo),intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.),intra-arterial, intramedulary, intracardiac, intra-articular (joint),intrasynovial (joint fluid area), intracranial, intraspinal, andintrathecal (spinal fluids). Any known device useful for parenteralinjection or infusion of pharmaceutical compositions can be used toeffect such administration.

Examples of parenteral dosage forms include aqueous solutions of theconjugates, in an isotonic saline, 5% glucose or other well-knownpharmaceutically-acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the dose of the conjugate. In one aspect of the presentembodiment, the conjugates may be formulated into prolonged or extendedrelease formulations such as, for example, the biodegradablecarbohydrate matrices described in U.S. Pat. Nos. 4,713,249, 5,266,333,and 5,417,982, the disclosures of each of which are incorporated hereinby reference in their entireties, or alternatively, a slow pump (e.g.,an osmotic pump) can be used.

In one aspect on the invention, at least one additional therapeuticfactor can be administered to a subject in combination with or as anadjuvant to the conjugates and methods of the present invention toenhance the conjugate-mediated elimination of the population ofpathogenic cells, or more than one therapeutic factor can be used. Inone example, the therapeutic factor can be selected from achemotherapeutic agent, or another therapeutic factor capable ofcomplementing the efficacy of the administered conjugate of the presentinvention. In another example, chemotherapeutic agents which are, forexample, cytotoxic themselves, can work to enhance tumor permeability,are also suitable for use as an additional therapeutic factor.

The therapeutic factor can be administered to the subject prior to,after, or at the same time as the conjugate, and the therapeutic factorcan be administered as part of the same composition containing theconjugate or as part of a different composition than the conjugate. Anysuch therapeutic composition containing the therapeutic factor at atherapeutically effective dose can be used in the present invention.

VI. Administration to Subjects A. Methods of Detection, Diagnosis andImaging

The amount of conjugate used in the methods of detection, diagnosis andimaging described herein will also depend on a variety of factors,including the identity of the targeting ligand, the identity of theimaging agent, the identity of the cancer or tumor to be detected, thelocation in the subject of the cancer or tumor, available means fordetecting the imaging agent, the means used to administer the conjugateto the subject, and the weight of the subject. Generally, the conjugateswill be administered to a subject in a method of detection, diagnosis orimaging in an amount of between about 0.1 mg to about 50 mg. Particularexamples include about 0.5 mg to about 10 mg, and from about 1 mg toabout 5 mg. In non-limiting examples, about 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg of a conjugate of the invention maybe a suitable amount of conjugate to be administered to a subject foruse in the methods of detection, diagnosis and imaging described herein.

The skilled artisan will understand that each of the methods ofdetection, diagnosis and imaging can be practiced using one type ofconjugate, or more than one type of conjugate, such as two, three, fouror more. When two or more types of conjugates are used, the methods canbe practiced by administering two or more types of conjugate to thesubject concurrently or sequentially, separated in time by 5, 10, 15,20, 25, 30 or more minutes, depending on the method being practiced.Illustratively, for example, the subject can be administered conjugateswith different targeting ligands, but the same active moiety in aco-dosing protocol. In other embodiments, the subject can beadministered conjugates comprising the same targeting ligand linked todifferent active moieties, or different targeting ligand linked todifferent active moieties.

When used in methods of detection, diagnosis and imaging, the conjugatesare preferably administered to a subject parenterally, e.g.,intradermally, subcutaneously, intramuscularly, intraperitoneally,intravenously, or intrathecally. However, the skilled artisan willunderstand that in some instances, administration may be oral,sublingual, intranasal, intraocular, rectal, transdermal, mucosal,pulmonary, or topical.

In addition to detecting the conjugates, the skilled artisan willunderstand that the amount of signal detected can be compared to othervalues, such as control values from a subject known to not have a tumoror cancer, or values obtained on an earlier date from the same subject.Thus, each of the methods of the present invention may alternativelycomprise measuring the amount of conjugate in the subject, rather thansimply detecting it, and optionally further comparing the measuredamount to a control value or a value obtained at an earlier time in thesame subject.

The means used to detect the conjugates vary based on factors includingthe identity of the imaging agent, whether the method is being practicedin vitro, in vivo or ex vivo, and when practiced in vivo, the locationin the subject to be visualized. Suitable means for detecting aconjugate comprising a radio-imaging agent include, but are not limitedto radioimaging systems, MRI, SPECT-CT, and PET imaging. Suitable meansfor detecting a conjugate comprising an optical imaging agent include,but are not limited to flow cytometry, confocal microscopy, fluorescenceactivated cell sorters, and fluorescence imaging systems such as LuminaII.

B. Methods of Treatment

The amount of conjugate used in the methods of treatment describedherein will also depend on a variety of factors, including the identityof the targeting ligand, the identity of the therapeutic agent, theidentity of the cancer or tumor to be detected, the location in thesubject of the cancer or tumor, the means used to administer theconjugate to the subject, and the health, age and weight of the subjectbeing treated. Generally, the conjugates will be administered to asubject in a method of treatment in an amount of between about 0.1 mg toabout 50 mg. Particular examples include about 0.5 mg to about 10 mg,and from about 1 mg to about 5 mg. In non-limiting examples, about 1 mg,2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg of a conjugateof the invention may be a suitable amount for administered to a subjectfor use in the methods of treatment described herein.

The skilled artisan will understand that the methods of treatment can bepracticed using one type of conjugate, or more than one type ofconjugate, such as two, three, four or more. When two or more types ofconjugates are used, the methods can be practiced by administering twoor more types conjugate to the subject concurrently or sequentially,separated in time by 5, 10, 15, 20, 25, 30 or more minutes, depending onthe method being practiced. Illustratively, for example, the subject canbe administered conjugates with different targeting ligands, but thesame active moiety in a co-dosing protocol. In other embodiments, thesubject can be administered conjugates comprising the same targetingligand linked to different active moieties, or different targetingligand linked to different active moieties.

Any effective regimen for administering the conjugates can be employed.Administration frequencies for a pharmaceutical composition comprisingone or more of the conjugates of the present invention include 4, 3, 2or once daily, every other day, every third day, every fourth day, everyfifth day, every sixth day, once weekly, every eight days, every ninedays, every ten days, bi-weekly, monthly and bi-monthly. The duration oftreatment will be based on the disease being treated and will be bestdetermined by the attending physician. However, continuation oftreatment is contemplated to last for a number of days, weeks, months oryears. Depending on the means of administration, a dosage may beadministered all at once, such as with an oral formulation in a capsuleor liquid, or slowly over a period of time, such as with anintramuscular or intravenous administration.

Illustratively, the conjugates can be administered as single doses, orcan be divided and administered as a multiple-dose daily regimen. Inother embodiments, a staggered regimen, for example, one to three daysper week can be used as an alternative to daily treatment, and for thepurpose of defining this invention such intermittent or staggered dailyregimen is considered to be equivalent to every day treatment and withinthe scope of this invention. According to at least one embodiment, thesubject is treated with multiple injections of the conjugate toeliminate a population of pathogenic cells. In another embodiment, thesubject is injected multiple times (preferably about 2 up to about 50times) with the conjugate, for example, at 12-72 hour intervals or at48-72 hour intervals. In other embodiments, additional injections of theconjugate can be administered to the subject at an interval of days ormonths after the initial injections(s) and the additional injectionsprevent recurrence of the disease state caused by the pathogenic cells.

When used in methods of treatment, the conjugates are preferablyadministered to a subject parenterally, e.g., intradermally,subcutaneously, intramuscularly, intraperitoneally, intravenously, orintrathecally. However, the skilled artisan will understand that in someinstances, administration may be oral, sublingual, intranasal,intraocular, rectal, transdermal, mucosal, pulmonary, or topical.

In relevant embodiments of the invention, the tumors and cancers thatcan be detected, diagnosed, imaged, and/or treated are any having cellsthat express CCK2R or CCK2i4svR, or both. In some instances, the tumorsand cancers will have cells that overexpress CCK2R or CCK2i4svR, orboth. The tumors and cancers include, but are not limited to, medullarythyroid cancers, insulinomas, small cell lung cancers, non small celllung cancers, astrocytoma, gastric cancer, bronchial and ilealcarcinoids, GIST tumors, and colon cancers, prostate cancer,hepatocellular carcinomas, and pancreatic cancers.

In each of the embodiments and aspects of the invention, the subject isa human, a non-human primate, bird, horse, cow, goat, sheep, a companionanimal, such as a dog, cat or rodent, or other mammal.

VII. Methods of Making the Conjugates

Generally, any manner of forming a conjugate between the linker and thetargeting ligand, and between the linker and the active moiety, can beutilized in accordance with the present invention. Also, anyart-recognized method of forming a conjugate between a spacer, acleavable linker, and one or more heteroatoms to form the linker L canbe used. The conjugate can be formed by direct conjugation of any ofthese molecules, for example, through hydrogen, ionic, or covalentbonds. Covalent bonding can occur, for example, through the formation ofamide, ester, disulfide, or imino bonds between acid, aldehyde, hydroxy,amino, sulfhydryl, or hydrazo groups.

Methods of synthesis are chosen depending upon the selection of theoptionally included heteroatoms or the heteroatoms that are alreadypresent on the spacers, cleavable linkers, targeting ligands, and activemoieties. In general, useful bond forming reactions are described inRichard C. Larock, “Comprehensive Organic Transformations, a guide tofunctional group preparations,” VCH Publishers, Inc. New York (1989),and in Theodora E. Greene & Peter G. M. Wuts, “Protective Groups ionOrganic Synthesis,” 2d edition, John Wiley & Sons, Inc. New York (1991),the disclosures of which are incorporated herein by reference in theirentireties.

More specifically, disulfide groups can be generally formed by reactingan alkyl or aryl sulfonylthioalkyl derivative, or the correspondingheteroaryldithioalkyl derivative such as a pyridin-2-yldithioalkylderivative, and the like, with an alkylenethiol derivative. For example,the required alkyl or aryl sulfonylthioalkyl derivative may be preparedaccording to the method of Ranasinghe and Fuchs, Synth. Commun. 18(3),227-32 (1988), the disclosure of which is incorporated herein byreference in its entirety. Other methods of preparing unsymmetricaldialkyl disulfides are based on a transthiolation of unsymmetricalheteroaryl-alkyl disulfides, such as 2-thiopyridinyl,3-nitro-2-thiopyridinyl, and like disulfides, with alkyl thiol, asdescribed in WO 88/01622, European Patent Application No. 0116208A1, andU.S. Pat. No. 4,691,024, the disclosures of which are incorporatedherein by reference in their entireties. Further, carbonates,thiocarbonates, and carbamates can generally be formed by reacting anhydroxy-substituted compound, a thio-substituted compound, or anamine-substituted compound, respectively, with an activatedalkoxycarbonyl derivative having a suitable leaving group.

VIII. Examples

The conjugates described herein may be prepared by conventional organicsynthesis methodology, or as otherwise indicated herein. Unless notedotherwise, all starting materials and reagents are generally availablethrough commercial suppliers.

A. Synthesis and Purification of Targeting Ligand Z-360³²⁻³⁵

Synthesis of targeting ligand Z-360 was accomplished according to thefollowing steps. Step 1—Boc D Diaminopropionic acid (3 g) was dissolvedin 100 ml of ethanol. Potassium carbonate (1.095 g) and2-fluoronitrobenzene (1.71 ml) was added followed by refluxing for 4hours. The reaction mixture was concentrated under pressure, water wasadded and the resulting mixture was washed with diethyl ether. 1 N HClwas the added to the aqueous layer to adjust its pH to 3 followed byextraction with ethyl acetate. The organic layer was then dried oversodium sulfate and then the solvent was evaporated under reducedpressure, where 4.5 g (95%) of the following compound was obtained as abright orange solid.

Step 2—The above product was used without further purification. It wasdissolved in 50 ml methanol followed by addition of 10% palladium carbon(50 mg). The resulting solution was purged with nitrogen gas and thenstirred at room temperature under hydrogen gas for 3 hrs. The reactionmixture was then filtered under celite, and the fitrate was concentratedunder pressure whereby 2-tert-butoxycarbonyl amino3(2-aminophenyl)-amino propionic acid was obtained as a dark browncompound. This compound was resuspended in toluene (120 ml) and theresulting suspension refluxed in toluene for 3 hrs overnight whileremoving water with a dean stark extractor. The solution was thenconcentrated under reduced pressure and purified by columnchromatography (ethyl acetate hexane) to obtain 1.722 g of the followingcompound as a mustard yellow solid. (Yield approximately 44%).

Step 3—To a solution of 1.722 g of the cyclized product(R)-(+)-2-oxo-3-tert-butoxycarbonylamino-1,3,4,5-tetrahydro-2H-1,5-benzodiazepine,in 13.7 ml of acetic acid were added 2.315 ml of cyclohexanone and 49 mgof platinum oxide. The resulting mixture was stirred at room temperaturefor 6 hrs under hydrogen atmosphere. To the reaction mixture was added 7ml of ethyl acetate and 25 mg of activated charcoal followed by stiflingfor an additional one hour at room temperature. The reaction mixture wasthe filtered, a 2 N aqueous solution of NaOH was added to the filtrateto neutralize the same under stifling after which it was separated intolayers. The organic layer was successively washed with a saturatedsolution of sodium bicarbonate and brine dried over anhydrous sodiumsulfate and then purified by column chromatography (ethylacetate/hexane) to obtain 1.0109 g of the following product(R)-(−)-2-oxo-3-tert-butoxycarbonylamino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepineas a pale yellow solid. (Yield approximately 45%).

Step 4—To a solution of(R)-(−)-2-oxo-3-tert-butoxycarbonylamino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepine(1.7131 g) in 6.8 ml anhydrous DMSO was added to 1.066 ml1-chloropinacolone and 0.9849 g of potassium carbonate, 0.039 potassiumiodide and 0.0459 g of tetrabutyl ammonium bromide. The solution wasstirred at 60° c. for 4 hrs. The reaction was quenched with water andthen the organic layer was washed with a saturated solution of sodiumbicarbonate and brine, dried over anhydrous sodium sulfate and thenpurified by column chromatography where 1.355 g of the following product(R)-1-tert-butylcarbonylmethyl-2-oxo-3-tert-butoxycarbonylamino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepinewere obtained as a yellow-brown solid. (Yield approximately 62%).

Step 5—To 1.355 g of the Boc-protected(R)-1-tert-butylcarbonylmethyl-2-oxo-3-tert-butoxycarbonylamino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepinewas added 2.2 ml 6 N concentrated hydrochloric acid and 2.2 ml ethanol.The mixture was stirred at 60 degrees for 2 hours. After the reactionmixture was cooled to room temperature, a mixed solvent of water anddiethyl ether (1:1) was added. The aqueous layer was separatelyneutralized with 6 N aqueous NaOH and then extracted with ethyl acetate.The extract was washed with brine, dried over anhydrous sodium sulfateand then evaporated under reduced pressure to yield 0.559 g. Thisproduct was further dissolved in 6.64 ml ethyl acetate and then to theresulting solution was added 0.197 oxalic acid dihydrate and then 4.4 mlhexane and the mixture was stirred overnight and allowed to crystallizeyielding 0.6790 g of(R)-(−)-1-tert-butylcarbonylmethyl-2-oxo-3-amino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepineoxalate monohydrate and(R)-(−)-1-tert-butylcarbonylmethyl-2-oxo-3-amino-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepineoxalate. (Yield approximately 51%).

Step 6—2.619 g of 3-amino benzoic acid was dissolved in 38.2 ml of 0.5 NNaOH followed by dropwise addition of a solution of 2.411 ml phenylchloroformate in 6.712 ml of THF. The reaction mixture was stirred forone hour. The precipitate that formed was then filtered with suction,washed with water, dried and then recrystallized from ethanol to obtaina white powder of 3-phenyloxycarbonylaminobenzoic acid. (Yieldapproximately 85%).

Step 7—Target LigandZ-360((R)-(−)-3-[3-(1-tert-butylcarbonylmethyl-2-oxo-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-3-yl)ureido]benzoicacid monohydrate). To a solution of 0.6790 g of the oxalate monohydratederivative above 7.290 ml anhydrous DMSO, 0.3819 g phenoloxy-(N-phenoxycarbonyl amino benzoic acid, 0.5915 ml triethylamine and catalyticamount of DMAP (5%). The resulting mixture was stirred at 60 degrees for2 hours and then 7.290 ml of ethanol were added to the reaction mixtureand the mixture was stirred at room temperature for an additional twohours. Under ice cooling, 7.290 ml of 1 N HCl was then added dropwise toprecipitate the following end product.

Stated another way, Z-360((R)-(−)-3-[3-(1-tert-butylcarbonylmethyl-2-oxo-5-cyclohexyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-3-yl)ureido]benzoicacid monohydrate) was synthesized as noted below in Scheme 1, wherein a)is potassium carbonate/ethanol, reflux 3 hrs; b) is H₂,10% Pd—C/methanol3 hrs; c) is toluene, reflux; d) is cyclohexanone, platinum oxide/aceticacid 6 h; e) is chloropinacolone, tetrabutyl ammonium bromide, potassiumcarbonate, potassium iodide, DMSO 4 h at 60 degrees; f) is 6 N HCl,Ethanol, 60 degrees, 2 h; g) is oxalic acid monohydrate/ethyl acetate,hexane; h) is phenoloxy(N-phenoxy-carbonyl amino benzoic acid, triethylamine DMAP DMSO; and i) is ethanol 2 h, 1 NHCl.

Referring to Scheme 1 above, the synthesis is as follows. The aromaticnucleophilic substitution reaction of 1-fluoro-2-nitrobenzene andBoc-(1)-2, 3, diaminopropionic acid in the presence of a base—potassiumcarbonate—in ethanol under refluxing conditions afforded compound 1 inexcellent yield. Reduction of the nitro group by catalytic hydrogenationled to compound 2. Compound 3 was formed in good yield by refluxingcompound 2 in toluene while removing water using a dean stark extractor.Subsequent reductive alkylation via catalytic hydrogenation in thepresence of acetic acid and Platinum oxide catalyst in a hydrogenatmosphere afforded the N5 regioselective compound 4. Regioselectivealkylation was again achieved by the reaction of chloropinacolone withcompound 5 in the presence of potassium iodide and tetrabutyl ammoniumbromide as a phase transfer catalyst to yield compound 5. Deprotectionof the N-tert butoxy carbonyl group was achieved by the use of a mixtureof hydrochloric acid and ethanol which yielded an amine hydrochloridesalt. This was then subjected to an anion exchange using oxalic aciddihydrate to yield compound 6. Compound 7, previously synthesized by thereaction of phenyl chloroformate and 3-aminobenzoic acid, was reactedwith compound 6 in the presence of anhydrous dimethylsulfoxide andtriethylamine. However this reaction proceeded very slowly and with pooryield. Addition of dimethylamino pyridine (DMAP) in catalytic amountsgreatly improved both the yield as well as the kinetics of the reaction.

B. Conjugates Comprising Z-360 and Radio-Imaging Agents

In order to determine the binding characteristics and specificity oftargeting ligands of interest, the following radio-imaging conjugateswere synthesized according to known methods³⁶, resulting in CCK2Rconjugates consisting of the targeting ligand and a Technetium ^(99m)(^(99m)Tc) chelating active moiety Dap-Asp-Cys, separated by the linkers(X) shown below.

CRL-1, where X=HN-Glu-Arg-Asp-COCRL-2, where X=HN-Glu-PS-Glu-PS-COCRL-3, where X=HN-Octanoyl-Glu-PS-Glu-PS-COCRL-4, where X=absent

As can be seen, CRL-1 contains a simple tripeptide spacer. Becausescavenger receptors in the kidneys and liver can bind diverse peptidicconjugates, the peptidic spacer in CRL-1 was replaced with a lessreadily scavenged peptidosaccharide (PS) spacer in CRL-2³⁷. Next, toassure sufficient separation between the targeting ligand and itstethered radiochelate, CRL-3 was designed with the same spacer as CRL-2,with an octanoyl moiety inserted before the peptidosaccharide spacer.Finally, to assist with evaluation of the impact of linker length, CRL-4was prepared with no linker. The detailed structure of each conjugatesis shown as follows.

The synthesis of the FMOC protected version of PS is provided asfollows. The sugar/peptidosaccharo monomer (PS) noted in the linkers ofCRL-2 and CRL-3 was synthesized according to Scheme 2 (below) usingpreviously described literature methods³⁷⁻³⁸. The monomer was used inthe synthesis of the linkers described for CRL-2 and CRL-3 above. Inshort, the free OH groups in D-glucamine were first protected usingdimethoxypropane in the presence of catalytic amounts ofp-toluenesulfonic acid. The amine group was then reacted withFmoc-glutamic-Oallyl to form an amide bond. The side chain carboxylicacid was deprotected using palladium tetrakis triphenylphosphine in thepresence of N-methyl morpholine acetic acid and chloroform. Thiscompound was used in solid phase synthesis in the same way amino acidmonomers were used. As shown in Scheme 2, to arrive at compound 16 (PS),the following reagents were used: a) p-TsOH, dimethoxypropane; b)acetone, dimethoxypropane, p-TsOH; c) Fmoc-glutamic-Oallyl, PYBOP,DIPEA/DMF; and d) Pd(PPh₃)₄, NMM/AcOH/CHCl₃.

Synthesis of CRL-3^(99m)Tc

CRL-3 ^(99m)Tc was produced by linking the targeting ligand Z-360 to a^(99m)Technetium chelating agent comprised of the peptide sequence:β-L-diaminopropionic acid (β-DAP), L-aspartic acid (L-Asp), L-cysteine(L-Cys) via linker shown in Scheme 3.

In particular, the ^(99m)TC chelating moiety (symbolized asEC20)(-DAP-Asp-Cys) was first prepared according to a reportedprocedure³⁶. Briefly, acid-sensitive Wang resin loaded with 0.106 mmolof H-carbonyl-trityl-L-cysteine (H-L-Cys (Trt)-OH) was reacted firstwith Fmoc-Asp(Otbu)-OH (0.265 mmol), HATU (0.265 mmol) anddiisopropylethylamine (1.06 mmol) followed by addition ofβ-L-diaminopropionic acid (0.265 mmol), HATU (0.265 mmol) anddiisopropylethylamine (1.06 mmol) to yield the ^(99m)Tc chelatingmoiety. The chelator was then conjugated to Z-360 via a variety ofspacers that were selected for both their abilities to render the finalconjugate water soluble and to reduce nonspecific binding to receptornegative cells. The monomeric components of these spacers were derivedfrom protected amino acids and a peptidosaccharide (PS) constructdescribed by others. All conjugation reactions were performed underargon atmosphere.acid-sensitive Wang resin loaded with 0.106 mmol offluorenylmethoxy carbonyl-trityl-L-cysteine (Fmoc-L-Cys(Trt)-OH) wasreacted first with HATU (0.265 mmol), followed by sequential addition ofthe desired protected monomer (0.265 mmol). Fmoc protecting groups wereremoved after each coupling step using standard conditions (20%piperidine in dimethyl formamide). Removal of the partially deprotectedconjugate from the polymeric support was finally accomplished bytreatment with a cocktail solution comprising of 92.5% trifluoroaceticacid (TFA), 2.5% 1,2-ethanedithiol, 2.5% triisopropylsilane, and 2.5%deionized water. This reaction also resulted in simultaneous removal ofall t-butyl (t-Bu), t-butoxycarbonyl (t-Boc) and trityl protectinggroups.

The crude product was purified by preparative reverse-phasehigh-performance liquid chromatography (RP-HPLC) using a Waters xTerraC18 10 μm; 19×250 mm column with a gradient mobile phase of A=20 mMammonium acetate buffer and B=acetonitrile; solvent gradient 5% B to 80%B in 30 minutes. Elution of the conjugate was monitored at k=280 nm andthe identities of the eluted compounds were analyzed by LC-MS and MALDI.Formulation and radiolabeling of the conjugates with ^(99m)Tc wasperformed according to previously described methods.

Formulation of the purified conjugate was accomplished in the followingmanner³⁶. A solution of stannous chloride dihydrate (0.8 mg, 0.003 mmol)in 0.02 M HCl (0.8 mL) was added to a solution of sodiumR-D-glucoheptonate dihydrate (800 mg, 2.815 mmol) in argon purged water(5.0 mL). Requisite peptide (0.001 mmol) was then added to the reactionmixture while purging with argon. After adjusting the pH of the solutionto 6.8±0.2 using 0.1 N NaOH, argon purged water was added to achieve atotal volume 10.0 mL. The solution mixture was dispensed into 5 mL vials(1.0 mL/vial) under argon atmosphere and lyophilized for 36 h. The vialswere sealed under argon atmosphere to yield the nonradioactiveformulation kits, which were stored at −20° C. until use. A solution ofsodium pertechnetate ^(99m)Tc (1.0 mL, 15 mCi) was added to a vial,heated in a boiling water bath for 18 min, and then cooled to roomtemperature before use.

Chelation of ^(99m)Tc by the conjugate was achieved by injecting 1 ml of^(99m)Tc-labeled sodium pertechnetate (15 mCi) into the vial and heatingfor ˜18 min in a boiling water bath (Scheme 1). The solution was allowedto cool to room temperature and stored in the dark until use on the sameday. Following chelation to ^(99m)Tc, corresponding conjugates areprefixed with ^(99m)Tc to indicate radiolabeling.

C. Receptor Binding Studies Using Radio-Imaging Conjugates

CRL-1, CRL-2, CRL-3 and CRL-4 were chelated to ^(99m)Tc and tested forbinding using two transfected cell lines of HEK-CCK2R (HEK-293 CCK2R)and HEK-CCK2R splice cell line (HEK CCK2i4svR), obtained from Dr. MarkHellmich (University of Texas at Galveston). Cells were cultured inDulbecco's modified Eagles (Gibco) supplemented with 10% Fetal BovineSerum and G418 disulfate (SIGMA; 400 μg/ml), 1% penicillin/streptomycinat 37° C. in a humidified 95% air, 5% CO₂ atmosphere.

Competition studies were performed using 100 fold excess of free Z-360containing corresponding peptide linker and no chelating moiety. HEK 293CCK2R and HEK CCK2i4svR were seeded onto 24 well plates and allowed tobecome confluent over 48-72 hours. Spent medium in each well wasreplaced with 0.5 ml fresh media containing 0.5% BSA and increasingconcentrations of the test article. After incubating for 1 h at 37° C.cells were rinsed with incubation solution (2×1.0 ml) to remove anyunbound radioactivity. Cells were then resuspended on 0.5 ml 0.25 N NaOHand cell radioactivity was counted using a γ-counter. The dissociationconstant (K_(D)) was calculated using a plot of cell bound radioactivityversus the concentration of the radiotracer using Graphpad Prism 4 andassuming a non-cooperative single site binding equilibrium.

As shown in FIG. 1 and FIG. 2, each of the conjugates bound toreceptor-expressing cells with high affinities in the low nano molarrange (as further supported by the data displayed in Table 2).

TABLE 2 Summary of binding affinity (Kd in nM) for radio- imaging^(99m)Tc conjugates of CRL-1, 2, 3, and 4 Kd (nM) HEK-293- Kd (nM)HEK-293- Conjugate No. CCK2R Cells CCKRi4svR Cells CRL-1 ^(99m)Tc 16 *CRL-2 ^(99m)Tc 46 31 CRL-3 ^(99m)Tc 30 4 CRL-4 ^(99m)Tc 270 * *K_(d)could not be determined due to high levels of non-specific binding.

As shown in FIG. 1 and FIG. 2, the solid line represents the cell boundradioactivity in the experimental group while the dashed line representsthe binding in presence of free Z-360. As can be seen, the inclusion ofthe peptidosaccharo monomer 16 (PS) was found to increase the watersolubility of the overall conjugate resulting in significantly higherspecificity as seen in the comparison between competition for CRL-1^(99m)Tc and CRL-2 ^(99m)Tc. However this also resulted in a decrease inthe binding affinity of conjugate CRL-2 ^(99m)Tc which may be attributedto steric effects from the bulkiness of the PS interfering with ligandbinding. Addition of an octanoic acid spacer between the PS monomers andthe targeting ligand resulted in an improvement in binding affinity forCRL-3 ^(99m)Tc. As a result of high affinity and specificity in receptorexpressing cells, CRL-3 ^(99m)Tc was used for subsequent radio-imagingexperiments in vivo.

D. In Vivo Radio-Imaging and Analysis of Biodistribution In VivoRadio-Imaging

In order to test the in vivo specificity of CRL-3 chelated with^(99m)Tc, HEK CCK2R and CCK2i4svR subcutaneous tumors were produced innude mice according to known methods. Athymic female nu/nu mice werepurchased from Harlan Laboratories and maintained on normal rodent chowand housed in a sterile environment on a standard 12 hour light and darkcycle for the duration of the study. All animal procedures were approvedby the Purdue Animal Care and Use Committee in accordance with NIHguidelines.

150 microcurie of CRL-3 ^(99m)Tc was injected intravenously into eachmouse. FIGS. 3A-B show an overlay of whole-body radio-images on whitelight photographs of mice bearing images of HEK CCK2R (a, b) and HEKCCKRi4sv (c, d) tumor xenografts in nu/nu mice two hours after theadministration of CRL-3 ^(99m)Tc. Kidneys were shielded with a leadplate. Mice a and c had the radioactive conjugate administered alongwith 100 fold of free unlabeled targeting ligand. In mice b and d, onlythe radio-conjugate CRL-3 ^(99m)Tc was administered. Arrows indicatelocation of the tumor.

As shown in FIGS. 3A-B, and specifically in mice b and d shown therein,the radio-conjugate accumulated mainly in the receptor expressing tumorwith little or no accumulation in other tissues except the kidney,signifying exceptional specificity to the tumor. Additionally, as shownin mice a and c, each of which were pre-injected with excess un-labeledtargeting ligand to block the receptors, the in vivo specificity of theradiolabeled conjugate was significant.

Bio-Distribution

Following the above radio-imaging studies, animals were dissected andselected organs/tissues were collected into pre-weighed γ-counter tubes.Radioactivity of weighed tissues and test compounds were counted in aγ-counter. CPM values were decay corrected and results were calculatedas % injected dose per gram of wet tissue (% ID/g). FIGS. 4A-B show thebio-distribution of the CRL-3 ^(99m)Tc in mice having HEK CCK2i4svR andHEK CCK2R tumors. Error bars represent the standard deviation (n=5mice/group). As will be appreciated, only the tumor and kidneys showexcessive accumulation of radiotracer in mice with HEK CCK2R and HEKCCK2i4svR tumors.

Time Course Bio-Distribution

Since small molecules are often excreted via the kidneys⁴³, we nextelected to determine whether uptake of CRL-3 ^(99m)Tc in the kidneysmight be transient. As seen in the SPECT-CT images of FIG. 5, the CRL-3^(99m)Tc content of the kidneys decreased significantly over time,whereas uptake in the tumor mass was relatively stable. By 24 h posttail vein injection, tumor to tissue ratios of CRL-3 ^(99m)Tc in themuscle, heart, skin, blood, liver, and spleen were 90, 83, 30, 61, 4 and14, respectively (see also Table 3). These data suggest that only CCK2Rpositive tissues retain the CRL-3 ^(99m)Tc, and therefore, thatradiation damage to normal tissues should be minimal.

TABLE 3 Tissue distribution of CRL-3 ^(99m)Tc in mice with subcutaneousHEK 293 -CCK2R cells at 0.5, 2, 4, 8 and 24 h post injection. Uptake ofradioactivity is expressed as percent injected dose per gram of wettissue. (n = 5 for hours 0.5, 4, 8, and 24; n = 10 for hour 2 timepoint). Organ 0.5 h 2 h 4 h 8 h 24 h Blood 5.8 ± 2.7  1.9 ± 0.97 1.7 ±0.39 0.24 ± 0.19  0.10 ± 0.062 Skin 2.3 ± 1.2  1.0 ± 0.41 1.1 ± 0.230.42 ± 0.22 0.21 ± 0.13 Spleen 2.1 ± 1.2 0.69 ± 0.70 1.4 ± 0.41 0.65 ±0.42 0.45 ± 0.39 Pancreas  1.3 ± 0.62 0.69 ± 0.84 0.29 ± 0.19   0.10 ±0.065 0.053 ± 0.031 Small  1.2 ± 0.63 0.52 ± 0.28 0.52 ± 0.085 0.19 ±0.10  0.13 ± 0.081 Intestine Large  1.0 ± 0.52 0.46 ± 0.20 0.53 ± 0.11 0.24 ± 0.16  0.13 ± 0.086 Intestine Stomach  1.2 ± 0.61 0.58 ± 0.32 0.56± 0.16  0.24 ± 0.15  0.15 ± 0.088 Liver 6.7 ± 3.6 2.5 ± 2.4 4.1 ± 0.641.9 ± 1.2 1.4 ± 1.4 Left 11.2 ± 6.1  8.4 ± 4.6 13.4 ± 2.1  6.0 ± 3.1 2.6± 1.6 Kidney Right 11.4 ± 6.3  7.6 ± 4.4 12.8 ± 1.8  5.7 ± 3.0 2.6 ± 1.7Kidney Heart 2.3 ± 1.5 0.68 ± 0.47 0.59 ± 0.18   0.14 ± 0.078 0.076 ±0.052 Lungs 3.2 ± 1.7  1.2 ± 0.55 1.0 ± 0.23 0.29 ± 0.15 0.18 ± 0.12Muscle  0.8 ± 0.37  0.29 ± 0.096 0.3 ± 0.04 0.093 ± 0.048 0.070 ± 0.080Brain  0.13 ± 0.072 0.051 ± 0.019 0.053 ± 0.016  0.064 ± 0.12  0.0076 ±0.0053 Tumor 7.4 ± 4.6 8.1 ± 5.1 12.0 ± 2.0  8.5 ± 4.9 6.3 ± 3.7

E. Conjugates Comprising Z-360 and Optical Imaging Agents

Given the specificity of Z-360 in targeting CCK2R expressing tumors,targeted optical imaging agents were developed and tested. Targeted,fluorescently-tagged imaging conjugates have the unique capability ofbeing used as a diagnostic agent as well as a tool to guide surgeonsduring inter-operative surgery for the removal of malignant tissue. Tothis end, three exemplary optical dyes (fluoresceine, rhodamine NHSester, and LS-288) were linked to Z-360. The structure of the resultingconjugates 17-19 are shown below:

As shown above, conjugate 17 correlates to Z-360 linked toFITC(CRL-FITC), i.e., having fluoresceine isothiocyanate conjugatedthereto; conjugate 18 correlates to Z-360 linked to rhodamine(CRL-Rhodamine), i.e., having rhodamine NHS ester conjugated thereto;conjugate 19 correlates to Z-360 linked to LS288 (CRL-LS288), i.e.,having LS-288-COOH conjugated thereto.

Synthesis of CRL

To prepare the optical imaging conjugates, the targeting ligand Z-360was attached to a tetrapeptide sequence: glutamic acid-arginine-asparticacid-lysine to a Cholecystokinin Receptor Ligand (CRL). Z-360 wassynthesized as described above (see, e.g., Scheme 1). The peptide spacerwas synthesized as follows. H-Lys (Boc)-2-C1-Trt resin (100 mg, 0.075mmol) was swollen in dichloromethane (2×5 ml) while bubbling underargon. A solution of Fmoc-Asp(OtBu)-OH (2.5 eq), HATU (2.5 eq), andDIPEA (5 eq) in DMF was added. The resulting solution was bubbled underargon for 4 hours, drained, and the resin washed with DMF (3×5 ml) andi-PrOH (3×5 ml). Fmoc deprotection was carried out using 20% piperidinein DMF (3×10 ml). The deprotection solution was removed, and the resinwashed again with DMF (3×5 ml) and i-PrOH (3×5 ml). Kaiser tests wereconducted to assess coupling and deprotection. The next amino acid inthe spacer, Fmoc-Arg(pbf)-OH, HATU, and DIPEA were then added in theamounts described above, and this procedure was repeated until all theamino acids and Z-360 (1.5 eq) were coupled to the growing spacer on theresin. The resin was washed with DMF (3×5 ml) and i-PrOH (3×5 ml),dichloromethane (2×5 ml), acetic acid (1×5 ml), and MeOH (1×5 ml), andallowed to dry under nitrogen. The peptide was then cleaved from theresin using a mixture of 95% TFA, 2.5% H₂O, and 2.5% triisopropylsilane.The solution was bubbled twice under nitrogen for 15 minutes, drained,concentrated, and then precipitated by addition of cold diethyl ether.Crude product was collected by centrifugation, washed three more timeswith diethyl ether, dried under vacuum and then purified by preparativereverse phase HPLC (Waters, xTerra C₁₈ 10 μm; 19×250 mm column mobilephase A=20 mM ammonium acetate buffer, pH 7, B-acetonitrile, gradient0-50% B in 30 minutes 13 ml/min X=280 nm). Pure fractions were analyzedby LC-MS and HR-MS pooled, and lyophilized to furnish CRL (compound 1 inScheme 4 below).

Scheme 4: Reagents and conditions for synthesis of CRL: (a) (i) DCM wash(ii) Fmoc-Asp(OtBu)-OH, HATU, DMF/DIPEA, 4 h. (b) (i) 20%piperidine/DMF, 30 min; (ii) Fmoc-Arg(pbf)-OH, HATU, DMF/DIPEA, 4 h. (c)(i) 20% piperidine/DMF, 30 min; (ii) Fmoc-Glu(OtBU)-OH, HATU, DMF/DIPEA,4 h. (d) (i) 20% piperidine/DMF, 30 min; (ii) Z-360, HATU, DMF/DIPEA, 4h. (e) TFA/H₂O/TIPS (95:2.5:2.5), 30 mins.

Synthesis of Conjugate 19

One equivalent of LS-288 COOH was dissolved in anhydrous DMSO (100 μL)containing five equivalent of diisopropylethylamine, and one equivalentof HATU was allowed to stir for 25 minutes under argon atmosphere. Athreefold molar excess of CRL was added and stirred at room temperaturefor overnight as outlined in Scheme 5. The product was then precipitatedby addition of isopropanol followed by centrifugation. The precipitatewas redissolved in DMSO and the crude products were purified bypreparative RP-HPLC using a Waters, xTerra C18 10 μm; 19×250 mm columnwith a gradient mobile phase of A=20 Mm ammonium acetate buffer;B=Acetonitrile; λ=280 nm; solvent gradient 5% B to 80% B in 30 minutes.Pure fractions were analyzed by LC-MS and LR-MS pooled and lyophilizedto furnish conjugate 19 (compound 3 (CRL-LS288) in Scheme 5 below).

Scheme 5: Reagents and conditions for synthesis of conjugate 19(CRL-LS288): (a) (i) HATU, DIPEA, DMSO, r.t. 25 mins, (ii) CRL spacer (3eq) stir overnight (iii) isopropanol, centrifugation.

Starting materials of 4 mg of Dye (LS288-COOH), 1.46 mg HATU, and 4.5 mgof peptide yielded 1.2 mg of final conjugate (yield approximately 15%).The remaining dye conjugates (conjugates 17 and 18) were synthesized ina method analogous to the one described above.

F. Receptor Binding Studies Using Optical Imaging Conjugates

FIGS. 6A-B display the results of binding by conjugate 18(CRL-Rhodamine) to HEK CCK2R cells as displayed in plates a-d in FIG.6A, and HEK CCKi4svR cells as displayed in plates e-h in FIG. 6B.Specifically, plates a and e show the binding of 10 nM conjugate 18;plates b and f show zoom in of the binding of 10 nM conjugate 18; platesc and g show the binding of 10 nM CRL-Rhodamine+excess competitor transillumination; and plates d and h show the binding of 10 nM conjugate18+excess competitor. Plates d and h show that all fluorescence isabolished when the CCK2R receptors are pre-blocked with CRL indicatingthat the fluorescent conjugates bind specifically via the CCK2 receptor.

FIGS. 7A-B display the binding of conjugate 19 (CRL-LS288) using twotransfected cell lines of HEK-CCK2R (HEK-293 CCK2R) and HEK-CCK2R splicecell line (HEK CCKi4svR), obtained from Dr. Mark Hellmich (University ofTexas at Galveston). Competition studies were performed using 100 foldexcess of free Z-360 containing corresponding peptide linker and nofluorescent dye. The resulting binding affinities are shown in FIGS. 7Aand 7B. As shown in each of FIGS. 7A-B, the solid line represents thecell-bound fluorescence in the experimental group while the dashed linerepresents the binding in presence of free Z-360.

FIGS. 8A-C display the binding of conjugate 19 in CCK2R tumors from micehaving HEK-CCK2R (HEK-293 CCK2R) tumors. Mouse a was pre-injected withexcess unlabeled Z-360-peptidoglycan conjugate prior to treating withconjugate 19, while mouse b was injected with 10 nmol of conjugate 19.Tumors and kidneys are shown with arrows. Dissected organs are labeledas follows: a-tumor, b-heart, c-lungs, d-spleen, e-liver, f-pancreasg-kidneys, h-small intestines, i-stomach.

FIGS. 9A-D display the binding of conjugate 19 in CCK2i4svR tumors frommice having HEK-CCK2R splice cell line (HEK CCK2i4svR) tumors. Mouse awas injected with 10 nmol of conjugate 19, while mouse b waspre-injected with excess unlabeled Z-360-peptidoglycan conjugate priorto injection with 10 nmol of conjugate 19, and mouse c containing areceptor negative KB tumor was also injected with 10 nmol conjugate 19.Tumors and kidneys are shown with. Dissected organs are labeled asfollows a-tumor, b-heart, c-lungs, d-spleen, e-liver, f-pancreasg-kidneys, h-small intestines, i-stomach.

Imaging CCK2R-Positive Metastasis Using Conjugate 19

In order to determine if conjugate 19 would be useful in identifyingmetastatic disease, it was used for imaging a murine metastasis model.Metastases were induced by injecting HEK-CCK2i4svR cells into theperitoneum of nu/nu mice and allowing the tumors to attach andproliferate for 3 weeks. Then, following tail vein injection of 10 nmolconjugate 19, mice were imaged both before and after various stages oftumor resection. As seen in FIG. 10 a, large tumor nodules could bereadily detected in the intact mice; i.e. prior to removal of skin andunderlying tissue. In fact, validation that the fluorescent spots seenin the intact mice accurately revealed the locations tumor noduleswithin the peritoneum was confirmed by removing the occluding skin andperitoneal lining and then re-imaging the animals (FIG. 10 b).Sequential fluorescence-guided surgical removal of the remainingfluorescent masses then yielded the images shown in FIGS. 10 c and 10 d.

Because of the density of the skin and bones covering the thoraciccavity, lung metastases could not be seen in the intact animals.However, following removal of the rib cage, numerous fluorescent lociwere revealed that appeared potentially malignant (FIG. 10 c & FIG. 11).To confirm that these fluorescent masses were indeed malignant,fluorescent tissues from both the thoracic and peritoneal cavities weresubmitted to pathology for hematoxylin and eosin staining. As shown inrepresentative samples in FIG. 12, all lesions that appeared fluorescentproved to be malignant (see arrows), indicating that CRL-LS288accurately identifies cancer metastases in murine models of malignantdisease.

G. Conjugates Comprising Z-360 and Therapeutic Agents

Given the specificity of Z-360 in targeting CCK2R-expressing tumors,even when bound to optical and radio-imaging reagents, conjugatescomprising therapeutic agents (tubulysine B hydrazide (TubH; compound20) and desacetyl vinblastine hydrazide (DAVBH; compound 21)) and weredeveloped and tested for efficacy.

Synthesis of Z-360-Linker B

Z-360 was first attached to a linker using the previously describedprocedure for the radio-imaging agent above to produce Z-360-linker B(Scheme 6 below).

Scheme 6: Synthesis of Z-360-linker B. Reagents and conditions: (a) (i)20% piperidine/DMF, rt, min; (ii)3,4,5,6-Di-isopropylidene-1-amino-deoxy(Fmoc-Glu-OH)-D-glucitol, HATU,DIPEA, 4 h; (b) (i) 20% piperidine/DMF, rt, 10 min; (ii)Fmoc-Glu(OtBu)-OH, HATU, DIPEA, 4 h; (c) (i) 20% piperidine/DMF, rt, 10min;(ii)3,4,5,6-Di-isopropylidene-1-amino-deoxy(Fmoc-Glu-OH)-D-glucitol,HATU, DIPEA, 4 h; (d) (i) 20% piperidine/DMF, rt, 10 min; (ii)Fmoc-Glu(OtBu)-OH, HATU, DIPEA, 4 h; (e)(i) 20% piperidine/DMF, rt, 10min; (ii) Fmoc-8-amino-octanoic, HATU, DIPEA, 4 h; (f) (i) 20%piperidine/DMF, rt, 10 min; Z-360, HATU, DIPEA, Overnight; i)TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5).

Synthesis of Conjugates Comprising Z-360 Linked to Tubulysin B Hydrazide(Compound 22; CRL-TubH)

Disulfide activated prodrugs (tubulysin B hydrazide³⁹ and desacetylvinblastine hydrazide⁴⁰) were synthesized according to literaturereported procedures.

Into a solution of saturated sodium bicarbonate (2 mL) and HPLC gradewater, argon was bubbled for 10 mM. With continuous bubbling of argon,Z-360-linker B (36 mg, 0.0226 mmol) was dissolved in argon purged HPLCgrade water (2.0 mL) and pH of the reaction mixture was increased to ˜7using argon purged bicarbonate. A solution of disulfideactivate-tubulysin hydrazide (12.0 mg, 0.0113 mmol) in THF (2.0 mL) wasthen added to the reaction mixture. After stirring for 20 mM, theprogress of the reaction was monitored using analytical RP-HPLC. At thispoint HPLC indicated that reaction was completed. After removing THFunder reduced pressure, Z-360-linker B-TubH (conjugate 22) was purifiedon a preparative RP-HPLC [A=2 mM ammonium acetate buffer (pH=7.0),B=CH₃CN, solvent gradient: 5% B to 80% B in 25 min] to yield requisiteproduct LRMS-LC/MS (m/z): (M+H)+calcd. for, C117H177N19038S3, 2553.96.found, 2554.

Scheme 7: Synthesis of conjugate 22. Reagents and conditions (a) i)H₂O/NaHCO₃ (pH=7.0-7.2), Argon, r.t.; ii) disulfide activated tubulysinB/THF, argon, r.t., 15 mins.

Synthesis of Conjugates Comprising Z-360 Linked to Desacetyl VinblastineHydrazide (Conjugate 23; CRL-DAVBH)

Following a similar procedure as described for preparation of conjugate22, desacetyl vinblastine hydrazide was synthesized from activatedvinblastine hydrazide. Z-360-linker B-desacetyl vinblastine hydrazide(conjugate 23) was purified by preparative RP-HPLC [A=20 mM ammoniumacetate (pH=7.2), B=CH₃CN, solvent gradient: 5% B to 80% B in 30 min],yielding the desired product LRMS (LC/MS) (m/z): calculated forC118H168N18036S2 2478 (M+H)⁺. Found 2478.

Synthesis of Untargeted Tubulysin Hydrazide and Untargeted DesacetylVinblastine Hydrazide

Following a similar procedure for preparation of conjugate 22, compoundscomprising therapeutic agents that lacked the targeting ligand wereproduced. Untargeted tubulysin hydrazide (Conjugate 24; Unt-TubH) anduntargeted desacetyl vinblastine hydrazide (Conjugate 25; Unt-DAVBH)were synthesized from activated TubH and DAVBH, respectively. Each ofthese compounds were purified by reverse phase HPLC [A=2 mM ammoniumacetate buffer (pH=7.0), B=CH₃CN, solvent gradient: 5% B to 80% B in 25min] to yield requisite product LRMS-LC/MS (m/z): (M+H)⁺ calcd. ForUnt-DAVBH, C₈₉H₁₃₄N₁₄O₃₂S₂, 1976.22. found, 1976 (m/z): (M+H)⁺ calcd.For Unt-TubH C₈₈H₁₄₃N₁₅O₃₄S₃, 2051.35. Found 2051.

Determination of In Vitro Potency of Targeted Therapeutic Agents

FIGS. 13A-C show the results of a study on the cytotoxicity of conjugate23 in HEK CCK2R cells, shown at varying hours from introduction. CW-2478denotes conjugate 23, with FIG. 13A showing values at 2 hours, FIG. 13Bshowing the values at 4 hours, and FIG. 13C showing the values at 9hours.

In further studies shown in FIGS. 14 and 15, vinblastine and tubulysin bagain exhibit dose dependent toxicity in HEK 293 cells expressing theCCK2R resulting in IC₅₀ of 8 and 1.3 nM respectively. Conjugates 22 and23 were included in these studies and both were found to be potent inHEK 293 cells expressing the CCK2R receptor with IC₅₀ of 12 and 28 nMrespectively (FIG. 14, 15). This indicates that conjugation with thelinker does not alter the intrinsic activity of the drugs. On the otherhand, Z-360 (FIG. 16) shows no concentration dependent cell toxicity forthe concentration range used. This may partly explain the modest in vivoantitumor observed activity despite its impressive antagonistic effects.The un-targeted drugs (Unt-TubH and Unt-DAVBH; FIG. 16), show a littleto no potency.

Determination of In Vivo Potency of Targeted Therapeutic Agents

Following the promising results obtained in vitro with the Z-360targeted chemotherapeutics, antitumor effects of the compounds wereinvestigated in vivo. HEK 293 cells expressing CCK2R (5.0×10⁶ in 50% HCmatrigel) were injected in the shoulder of 5-6 week old female nu/numice. Tumors were measured in two perpendicular directions every two tothree days with vernier calipers, and their volumes calculated as0.5×L×W² where L is the longest axis (in millimeters) and W is the axisperpendicular to L (in millimeters). Dosing solutions were prepared insaline and administered through either the lateral vein of the mice forTubulysin B conjugates or intraperitoneally for desacetylvinblastinehydrazide conjugates. Dosing was initiated when the subcutaneous tumorsreached ˜100 mm³ in volume. Each mouse received 2 μmol/kg of the testconjugate in 100 microliters of saline per injection, three times a weekfor three weeks. Mouse weights were also recorded at each dosing as ameasure of gross toxicity. Mice in the control groups received notreatment.

As shown in FIGS. 17 a and 19 a, in comparison to untreated controlsconjugates 22 and 23 showed remarkable antitumor activity in CCK2Rexpressing tumor bearing mice. However whereas conjugate 23-treatedtumors were observed to remain the same size during the period of thestudy, treatment with conjugate 22 resulted in reduction of tumor sizesand disappearance of ⅗ tumors. The remaining tumor masses from theconjugate 23 group were analyzed by H&E staining and as can be seen inFIG. 18 with the remaining mass in the conjugate 23 and conjugate23+competition groups contain very few malignant cells. As shown in FIG.17 a, tumors treated with the untargeted compounds (Unt-DAVBH) grew atalmost the same rate as untreated controls. On the other hand, theuntargeted tubulysin compound (Unt-TubH; FIG. 19 a) demonstrated modesttumor inhibition consistent with the partial potency observed in the invitro studies using the same compound. Importantly, body weights in eachof the groups (FIGS. 17 b and 19 b) studied remained essentiallyunchanged over the course of the study suggesting that targeted thetherapy is not grossly toxic to the animal.

Surprisingly, mice that were pretreated with 100-fold excess of Z-360with the aim of blocking the receptors, still exhibited antitumoractivity when treated with the targeted drugs. We hypothesize that thismay be due to the fact that once the conjugate localizes in the tumorcell surface the disulfide bond gets cleaved releasing free drug thatdiffuses through the membrane.

While the invention has been described with reference to certainparticular embodiments thereof, those skilled in the art will appreciatethat various modifications may be made without departing from the spiritand scope of the invention. The scope of the appended claims is not tobe limited to the specific embodiments described.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains. Each cited patent and publication isincorporated herein by reference in its entirety. All of the followingreferences have been cited in this application:

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What is claimed is:
 1. A conjugate of the following formula B-[L-D]_(n) wherein B is a targeting ligand of Formula I

wherein: the bond between X and Y is a single bond or a double bond, R¹=H, CH₃ or heterocycle with R²; R²=heterocycle with R¹, CH₃, CH₂CON(Et)₂, CH₂COC(CH₃)₃, CH₂CONHC(CH₃)₃,

X=CR³ or NR⁴; R³=phenyl, cyclohexyl, CH₃, CH₂—CH₃,

R⁴=phenyl, cyclohexyl,

Y=CH₂, N (dashed=double bond), C═O; z=

R⁵=absent, phenyl, C₁, CO₂H, CH₃, OCH₃, NHCH₃, F, SCH₂CO₂H, SCH₂CO₂Et,

and analogs, derivatives, salts and esters thereof, L is a bivalent or polyvalent linker, D is an active moiety, wherein each active moiety is individually selected from therapeutic agents and imaging agents, and n is an integer of between 1 and
 5. 2. The conjugate of claim 1, wherein the targeting ligand B is the benzodiazepine Z-360


3. The conjugate of claim 1, wherein each active moiety D is a therapeutic agent individually selected from a radio-therapeutic agent, immunotherapeutic agent, photodynamic therapy agent and chemotherapeutic agent.
 4. The conjugate of claim 1, wherein each active moiety D is an imaging agent individually selected from a radio-imaging agent, an optical imaging agent, a PET imaging agent, a MRI contrast agent, a CT contrast agent and a FRET imaging agent.
 5. The conjugate of claim 3, wherein the therapeutic agent is tubulysin B hydrazide or desacetyl vinblastine monohydrazide.
 6. The conjugate of claim 4, wherein imaging agent is fluorescein (FITC), rhodamine, S0456, IR800CW, or LS288.
 7. The conjugate of claim 1, wherein the linker L is L1, L2 or L3 L1: HN-Glu-Arg-Asp-CO L2: HN-Glu-PS-Glu-PS-CO L3: HN-Octanoyl-Glu-PS-Glu-PS-CO wherein PS is the following formula


8. The conjugate of claim 1, wherein n=1.
 9. A conjugate selected from the group consisting of CRL-1, CRL-2, CRL-3, CRL-4, conjugate 17, conjugate 18, conjugate 19, conjugate 22, and conjugate 23


10. A method for detecting a tumor in a subject, comprising administering a conjugate of claim 1 to a subject suspected of having a tumor and detecting the conjugate in the subject, wherein the active moiety D of the conjugate is an imaging agent.
 11. The method of claim 10, wherein the imaging agent is fluorescein (FITC), rhodamine, S0456, IR800CW, or LS288.
 12. The method of claim 10, wherein the imaging agent is radio-imaging agent comprising ¹¹¹In, ⁹⁹ mTc, ⁶⁴CU, ⁶⁷Cu, ⁶⁷Ga or ⁶⁸Ga.
 13. The method of claim 11 wherein the conjugate is detected utilizing flow cytometry, confocal microscopy, fluorescence activated cell sorters, or a fluorescence imaging system.
 14. The method of claim 12, wherein the conjugate is detected utilizing a radioimaging system, MRI, SPECT-CT, or PET imaging.
 15. A method for treating a subject having cancer, comprising administering a therapeutically-effective amount of a conjugate of claim 1 to a subject having cancer, wherein the active moiety D of the conjugate is a therapeutic agent.
 16. The method of claim 15, wherein the therapeutic agent is tubulysin B hydrazide or desacetyl vinblastine monohydrazide.
 17. The method of claim 15, wherein the conjugate is in a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable diluent or carrier.
 18. The method of claim 15, wherein the cancer is a cancer selected from the group consisting of medullary thyroid cancers, insulinomas, small cell lung cancers, non small cell lung cancers, astrocytoma, gastric cancer, bronchial and ileal carcinoids, GIST tumors, and colon cancers, prostate cancer, hepatocellular carcinomas, and pancreatic cancers. 